Differentiation of human embryonic stem cells

ABSTRACT

The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides an improved method for the formation of pancreatic endoderm, pancreatic hormone expressing cells and pancreatic hormone secreting cells. The present invention also provides methods to promote the differentiation of pluripotent stem cells without the use of a feeder cell layer.

FIELD OF THE INVENTION

The present invention provides methods to promote the differentiation ofpluripotent stem cells. In particular, the present invention provides animproved method for the formation of pancreatic endoderm, pancreatichormone expressing cells and pancreatic hormone secreting cells. Thepresent invention also provides methods to promote the differentiationof pluripotent stem cells without the use of a feeder cell layer.

BACKGROUND

Advances in cell-replacement therapy for Type I diabetes mellitus and ashortage of transplantable islets of Langerhans have focused interest ondeveloping sources of insulin-producing cells, or β cells, appropriatefor engraftment. One approach is the generation of functional β cellsfrom pluripotent stem cells, such as, for example, embryonic stem cells.

In vertebrate embryonic development, a pluripotent cell gives rise to agroup of cells comprising three germ layers (ectoderm, mesoderm, andendoderm) in a process known as gastrulation. Tissues such as, forexample, thyroid, thymus, pancreas, gut, and liver, will develop fromthe endoderm, via an intermediate stage. The intermediate stage in thisprocess is the formation of definitive endoderm. Definitive endodermcells express a number of markers, such as, HNF-3beta, GATA4, Mixl1,CXCR4 and Sox-17.

Formation of the pancreas arises from the differentiation of definitiveendoderm into pancreatic endoderm. Cells of the pancreatic endodermexpress the pancreatic-duodenal homeobox gene, Pdx1. In the absence ofPdx1, the pancreas fails to develop beyond the formation of ventral anddorsal buds. Thus, Pdx1 expression marks a critical step in pancreaticorganogenesis. The mature pancreas contains, among other cell types,exocrine tissue and endocrine tissue. Exocrine and endocrine tissuesarise from the differentiation of pancreatic endoderm.

Cells bearing the features of islet cells have reportedly been derivedfrom embryonic cells of the mouse. For example, Lumelsky et al. (Science292:1389, 2001) report differentiation of mouse embryonic stem cells toinsulin-secreting structures similar to pancreatic islets. Soria et al.(Diabetes 49:157, 2000) report that insulin-secreting cells derived frommouse embryonic stem cells normalize glycemia in streptozotocin-induceddiabetic mice.

In one example, Hori et al. (PNAS 99: 16105, 2002) disclose thattreatment of mouse embryonic stem cells with inhibitors ofphosphoinositide 3-kinase (LY294002) produced cells that resembled βcells.

In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports thegeneration of insulin-producing cells from mouse embryonic stem cellsconstitutively expressing Pax4.

Micallef et al. reports that retinoic acid can regulate the commitmentof embryonic stem cells to form Pdx1 positive pancreatic endoderm.Retinoic acid is most effective at inducing Pdx1 expression when addedto cultures at day 4 of embryonic stem cell differentiation during aperiod corresponding to the end of gastrulation in the embryo (Diabetes54:301, 2005).

Miyazaki et al. reports a mouse embryonic stem cell line over-expressingPdx1. Their results show that exogenous Pdx1 expression clearly enhancedthe expression of insulin, somatostatin, glucokinase, neurogenin3, P48,Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53:1030, 2004).

Skoudy et al. reports that activin A (a member of the TGFβ superfamily)upregulates the expression of exocrine pancreatic genes (p48 andamylase) and endocrine genes (Pdx1, insulin, and glucagon) in mouseembryonic stem cells.

The maximal effect was observed using 1 nM activin A. They also observedthat the expression level of insulin and Pdx1 mRNA was not affected byretinoic acid; however, 3 nM FGF7 treatment resulted in an increasedlevel of the transcript for Pdx1 (Biochem. J. 379: 749, 2004).

Shiraki et al. studied the effects of growth factors that specificallyenhance differentiation of embryonic stem cells into Pdx1 positivecells. They observed that TGFβ2 reproducibly yielded a higher proportionof Pdx1 positive cells (Genes Cells. 2005 June; 10(6): 503-16.).

Gordon et al. demonstrated the induction ofbrachyury+/HNF-3beta+endoderm cells from mouse embryonic stem cells inthe absence of serum and in the presence of activin along with aninhibitor of Wnt signaling (US 2006/0003446A1).

Gordon et al. (PNAS, Vol 103, page 16806, 2006) states “Wnt andTGF-beta/nodal/activin signaling simultaneously were required for thegeneration of the anterior primitive streak”.

However, the mouse model of embryonic stem cell development may notexactly mimic the developmental program in higher mammals, such as, forexample, humans.

Thomson et al. isolated embryonic stem cells from human blastocysts(Science 282:114, 1998). Concurrently, Gearhart and coworkers derivedhuman embryonic germ (hEG) cell lines from fetal gonadal tissue(Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlikemouse embryonic stem cells, which can be prevented from differentiatingsimply by culturing with Leukemia Inhibitory Factor (LIF), humanembryonic stem cells must be maintained under very special conditions(U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).

D'Amour et al. describes the production of enriched cultures of humanembryonic stem cell-derived definitive endoderm in the presence of ahigh concentration of activin and low serum (D'Amour K A et al. 2005).Transplanting these cells under the kidney capsule of mice resulted indifferentiation into more mature cells with characteristics of someendodermal organs. Human embryonic stem cell-derived definitive endodermcells can be further differentiated into Pdx1 positive cells afteraddition of FGF-10 (US 2005/0266554A1).

D'Amour et al. (Nature Biotechnology-24, 1392-1401 (2006)) states “Wehave developed a differentiation process that converts human embryonicstem (hES) cells to endocrine cells capable of synthesizing thepancreatic hormones insulin, glucagon, somatostatin, pancreaticpolypeptide and ghrelin. This process mimics in vivo pancreaticorganogenesis by directing cells through stages resembling definitiveendoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursoren route to cells that express endocrine hormones”.

In another example, Fisk et al. reports a system for producingpancreatic islet cells from human embryonic stem cells(US2006/0040387A1). In this case, the differentiation pathway wasdivided into three stages. Human embryonic stem cells were firstdifferentiated to endoderm using a combination of n-butyrate and activinA. The cells were then cultured with TGFβ antagonists such as Noggin incombination with EGF or betacellulin to generate Pdx1 positive cells.The terminal differentiation was induced by nicotinamide.

In one example, Benvenistry et al. states: “We conclude thatover-expression of Pdx1 enhanced expression of pancreatic enrichedgenes, induction of insulin expression may require additional signalsthat are only present in vivo” (Benvenistry et al, Stem Cells 2006;24:1923-1930).

Therefore, there still remains a significant need to develop conditionsfor establishing pluripotent stem cell lines that can be expanded toaddress the current clinical needs, while retaining the potential todifferentiate into pancreatic endocrine cells, pancreatic hormoneexpressing cells, or pancreatic hormone secreting cells. We have takenan alternative approach to improve the efficiency of differentiatinghuman embryonic stem cells toward pancreatic endocrine cells.

SUMMARY

In one embodiment, the present invention provides a method fordifferentiating pluripotent stem cells, comprising the steps of:

-   -   a. Culturing the pluripotent stem cells, and    -   b. Differentiating the pluripotent stem cells into cells        expressing markers characteristics of the definitive endoderm        lineage,    -   c. Differentiating the cells expressing markers characteristics        of the definitive endoderm lineage into cells expressing markers        characteristics of the pancreatic endoderm lineage, and    -   d. Differentiating the cells expressing markers characteristics        of the pancreatic endoderm lineage into cells expressing markers        characteristics of the pancreatic endocrine lineage.

In one embodiment, cells expressing markers characteristic of thedefinitive endoderm lineage are differentiated from pluripotent stemcells by treating pluripotent stem cells by any one of the followingmethods:

-   -   a. Culturing the pluripotent stem cells in medium containing        activin A in the absence of serum, then culturing the cells with        activin A and serum, and then culturing the cells with activin A        and serum of a different concentration, or    -   b. Culturing the pluripotent stem cells in medium containing        activin A in the absence of serum, then culturing the cells with        activin A with serum of another concentration, or    -   c. Culturing the pluripotent stem cells in medium containing        activin A and a Wnt ligand in the absence of serum, then        removing the Wnt ligand and culturing the cells with activin A        with serum, or    -   d. Culturing the pluripotent stem cells on a tissue culture        substrate coated with an extracellular matrix, and culturing the        pluripotent stem cells with activin A and a Wnt ligand, or    -   e. Culturing the pluripotent stem cells on a tissue culture        substrate coated with an extracellular matrix, then culturing        the pluripotent stem cells with activin A and a Wnt ligand in a        first culture medium containing serum, then culturing the        pluripotent stem cells with activin A in a second culture medium        containing serum, or    -   f. Culturing the pluripotent stem cells on a tissue culture        substrate coated with an extracellular matrix, then culturing        the pluripotent stem cells with activin A and a Wnt ligand in a        first culture medium containing serum, then culturing the        pluripotent stem cells with activin A and a Wnt ligand in a        second culture medium containing serum of a different        concentration.

In one embodiment, cells expressing markers characteristic of thepancreatic endoderm lineage are differentiated from cells expressingmarkers characteristic of the definitive endoderm lineage by treatingcells expressing markers characteristic of the definitive endodermlineage by any one of the following methods:

-   -   a. Treating the cells expressing markers characteristic of the        definitive endoderm lineage with a fibroblast growth factor and        a hedgehog signaling pathway inhibitor, then removing the medium        containing the fibroblast growth factor and the hedgehog        signaling pathway inhibitor and subsequently culturing the cells        in medium containing retinoic acid, a fibroblast growth factor        and the hedgehog signaling pathway inhibitor, or    -   b. Treating the cells expressing markers characteristic of the        definitive endoderm lineage with retinoic acid and at least one        fibroblast growth factor, or    -   c. Treating the cells expressing markers characteristic of the        definitive endoderm lineage with retinoic acid, then removing        the retinoic acid and subsequently treating the cells with at        least one fibroblast growth factor.

In one embodiment, cells expressing markers characteristic of thepancreatic endocrine lineage are differentiated from cells expressingmarkers characteristic of the pancreatic endoderm lineage by treatingcells expressing markers characteristic of the pancreatic endodermlineage by any one of the following methods:

-   -   a. Culturing the cells expressing markers characteristic of the        pancreatic endoderm lineage in medium containing DAPT and        exendin 4, then removing the medium containing DAPT and exendin        4 and subsequently culturing the cells in medium containing        exendin 1, IGF-1 and HGF, or    -   b. Culturing the cells expressing markers characteristic of the        pancreatic endoderm lineage in medium containing exendin 4, then        removing the medium containing exendin 4 and subsequently        culturing the cells in medium containing exendin 1, IGF-1 and        HGF, or    -   c. Culturing the cells expressing markers characteristic of the        pancreatic endoderm lineage in medium containing DAPT and        exendin 4, or    -   d. Culturing the cells expressing markers characteristic of the        pancreatic endoderm lineage in medium containing exendin 4, or    -   e. Treating the cells expressing markers characteristic of the        pancreatic endoderm lineage with a factor that inhibits the        Notch signaling pathway.

In one embodiment, the present invention provides a method for treatinga patient suffering from diabetes, comprising the steps of:

-   -   a. Culturing pluripotent stem cells,    -   b. Differentiating the pluripotent stem cells into cells        expressing markers characteristics of the definitive endoderm        lineage,    -   c. Differentiating the cells expressing markers characteristics        of the definitive endoderm lineage into cells expressing markers        characteristics of the pancreatic endoderm lineage,    -   d. Differentiating the cells expressing markers characteristics        of the pancreatic endoderm lineage into cells of a β-cell        lineage, and    -   e. Implanting the cells of a β-cell lineage into the patient.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the expression of the definitive endoderm markers CXCR4,GATA4, HNF-3beta, Mixl1, Sox-17 in the human embryonic stem cell line H9following treatment with 100 ng/ml activin A for two, five and eightdays. Expression of definitive endoderm markers was assayed at the mRNAlevel and normalized to expression levels in untreated human embryonicstem cells. FIG. 1B shows the expression of the anterior endodermmarkers Cerberus, Otx-1 and Hex genes in the human embryonic stem cellline H9 following treatment with 100 ng/ml activin A for three and fivedays.

FIGS. 2A, 2B and 2C shows the expression of definitive endoderm markersin the human embryonic stem cell line H9 following treatment with 100ng/ml activin A for five days. Expression of the definitive endodermmarkers was detected by immunohistochemistry. FIG. 2A shows Sox-17expression. FIG. 2B shows HNF-3beta expression. FIG. 2C shows Oct3/4expression.

FIGS. 3A, 3B, 3C and 3D show the expression of definitive endodermmarkers in the human embryonic stem cell line H9 following a step-wisedifferentiation protocol. Expression of the definitive endoderm markerswas assayed at the mRNA level and normalized to expression levels inuntreated human embryonic stem cells. FIG. 3A shows GATA4 expression.FIG. 3B shows Sox-17 expression. FIG. 3C shows HNF-3beta expression.

FIG. 3D shows Mixl1 expression. Data points marked ‘AA’ denote activin Atreatment for one (1d), three (3d), five (5d), or seven days (7d). Datapoints marked ‘UT’ denote untreated controls cultured for one (1d),three (3d), five (5d), or seven days (7d).

FIGS. 4A and 4B show the expression of extra-embryonic endoderm markersin the human embryonic stem cell line H9 following a step-wisedifferentiation protocol. Expression of the extraembryonic endodermmarkers was assayed at the mRNA level and normalized to expressionlevels in untreated human embryonic stem cells. FIG. 4A shows the effectof 100 ng/ml activin A on AFP expression. FIG. 4B shows the effect of100 ng/ml activin A on Sox7 expression. Data points marked ‘AA’ denoteactivin A treatment for one (1d), three (3d), five (5d), or seven days(7d). Data points marked ‘UT’ denote untreated controls cultured for one(1d), three (3d), five (5d), or seven days (7d).

FIGS. 5A and 5B show the expression of mesoderm and ectoderm markers inthe human embryonic stem cell line H9 following a step-wisedifferentiation protocol. Expression of the mesoderm and ectodermmarkers was assayed at the mRNA level and normalized to expressionlevels in untreated human embryonic stem cells. FIG. 5A shows the effectof 100 ng/ml activin A on Brachyury expression. FIG. 5B shows the effectof 100 ng/ml activin A on Zic1 expression. Data points marked ‘AA’denote activin A treatment for one (1d), three (3d), five (5d), or sevendays (7d). Data points marked ‘UT’ denote untreated controls culturedfor one (1d), three (3d), five (5d), or seven days (7d).

FIGS. 6A, 6B, 6C, 6D, 6E and 6F show the expression of the definitiveendoderm markers Brachyury (FIG. 6A) CXCR4 (FIG. 6B), Mixl1 (FIG. 6C),Sox17 (FIG. 6D), HNF-3beta (FIG. 6E), Oct4 (FIG. 6F) in the humanembryonic stem cell line H7 following treatment with 100 ng/ml activin Afor one, three, five and seven days. Expression of definitive endodermmarkers was assayed at the mRNA level and normalized to expressionlevels in untreated human embryonic stem cells.

FIGS. 7A, 7B, 7C, 7D, 7E and 7F shows the expression of definitiveendoderm markers in the human embryonic stem cell line H9 followingapplication of a differentiation protocol. Expression of the definitiveendoderm markers was detected by immunohistochemistry. FIGS. 7A and 7Bshow Sox-17 expression. FIGS. 7C and 7D show HNF-3beta expression. FIGS.7E and 7F show GATA4 expression. FIGS. 7B, 7D and 7F show counterstaining of the nuclei with DAPI. The columns marked ‘treated’ denoteactivin A treatment (100 ng/ml) for five days. The columns marked‘untreated’ denote untreated controls.

FIGS. 8A, 8B and 8C shows the expression of pancreatic endoderm markersin the human embryonic stem cell line H9 following application of asecond differentiation protocol. Expression of the pancreatic endodermmarkers was assayed by PCR and normalized to expression levels inactivin A treated human embryonic stem cells. FIG. 8A shows Pdx1expression. FIG. 8B shows GLUT-2 expression. FIG. 8C shows PTF1aexpression.

FIGS. 9A and 9B show the expression of pancreatic endoderm markers inthe human embryonic stem cell line H9 following application of a seconddifferentiation protocol. Expression of the pancreatic endoderm markerswas detected by immunohistochemistry. FIG. 9A shows Pdx1 expression inthe untreated control, and FIG. 9B shows Pdx1 expression in the culturetreated by the step-wise differentiation protocol.

FIGS. 10A, 10B, 10C and 10D show the expression of pancreatic endocrinemarkers in the human embryonic stem cell line H9 following applicationof a third differentiation protocol. Expression of the pancreaticendocrine markers was assayed by PCR and normalized to expression levelsin activin A treated human embryonic stem cells. FIG. 10A shows NeuroD1expression. Figure shows Ngn3 expression. FIG. 10C shows insulinexpression. FIG. 10D shows Hes-1 expression, the expression level isnormalized to pancreatic endoderm cells.

FIGS. 11A and 11B show the expression of pancreatic endoderm markers inthe human embryonic stem cell line H9 following application of adifferentiation protocol. Expression of the pancreatic endoderm markerswas assayed by PCR and normalized to expression levels in activin Atreated human embryonic stem cells. FIG. 11A shows Nkx2.2 expression.FIG. 11B shows Pdx1 expression.

FIG. 12 shows the expression of PDX-1 in cells with each passage (P0, P1and P2) in culture. Expression of the PDX-1 was assayed by PCR andnormalized to expression levels in activin A treated human embryonicstem cells H9.

FIGS. 13A and 13B show the expression of hepatocyte markers in the humanembryonic stem cell line H9 following application of a thirddifferentiation protocol. Expression of the hepatocyte markers wasassayed by PCR and normalized to expression levels in activin A treatedhuman embryonic stem cells. FIG. 13A shows AFP expression. FIG. 13Bshows albumin expression.

FIGS. 14A and 14B shows the expression of markers of pluripotency in thehuman embryonic stem cell line H9. Expression of the markers ofpluripotency was assayed by immunohistochemistry. FIG. 14A shows Oct-4expression. FIG. 14B shows alkaline phosphatase expression.

FIG. 15 shows the karyotype of the human embryonic cell line H9. TheKaryotype was determined on cells at passage number P36 that werecultured on mouse embryonic fibroblast feeder cells.

FIG. 16 depicts the outline of a differentiation protocol in thisinvention, where human embryonic stem cells are differentiated intodefinitive endoderm in a feeder free system.

FIGS. 17A, 17B, 17C, 17D, 17E and 17F depict the FACS profile of humanembryonic stem cell line H9 at passage number 44, cultured on varyingconcentrations of MATRIGEL and exposed to (0.5-2%) low serum and highactivin A (100 ng/ml) for 5 days. The expression of definite endodermmarker CXCR4 (CD184) is shown on the Y-axis and the expression of ESmarker CD9 is shown on the X-axis.

FIG. 18 shows the real-time PCR results for markers of definitiveendoderm, from cultures of the human embryonic stem cell line H9 atpassage 44 cultured on a 1:10 dilution of MATRIGEL (▪), a 1:20 dilutionof MATRIGEL (▪), or a 1:30 dilution of MATRIGEL (□) and exposed to thedifferentiation protocol disclosed in Example 14. The fold induction isrelative to undifferentiated cells of the human embryonic stem cell lineH9, at passage number 44, cultured in medium conditioned using mouseembryonic fibroblasts.

FIGS. 19A and 19B show the scatter plots for global gene expression inundifferentiated pluripotent stem cells and definitive endoderm cellsobtained from differentiating pluripotent stem cells. Data shown is fromcultures of the human embryonic stem cell line H9 cell line at passage44 cultured on mouse embryonic fibroblasts (right panel) and passage 83cultured on MATRIGEL (left panel).

FIGS. 20A, 20B and 20C depict the expression of CXCR4 by FACS at day 5for the human embryonic stem cell line H1 (FIG. 20A), the humanembryonic stem cell line H7 (FIG. 20B), and the human embryonic stemcell line H9 (FIG. 20C) cultured on mouse embryonic fibroblast feedercells exposed to the definitive endoderm differentiation protocoldisclosed in Example 4.

FIGS. 21A and 21B show the real-time PCR results of expression of theindicated definitive endoderm markers in cultures of the human embryonicstem cell line H7 (FIG. 21A) and the human embryonic stem cell line H9(FIG. 21B) cultured on mouse embryonic fibroblast feeder cells. Resultsare expressed as fold increase over undifferentiated cells.

FIGS. 22A, 22B and 22C depict the expression of CXCR4 by FACS at day 5for the human embryonic stem cell line H1 (FIG. 22A), the humanembryonic stem cell line H7 (FIG. 22B), and the human embryonic stemcell line H9 (FIG. 22C) cultured on MATRIGEL (1:30 dilution) and exposedto the definitive endoderm differentiation protocol disclosed in Example4.

FIGS. 23A, 23B and 23C show the real-time PCR results of the expressionof the indicated definitive endoderm markers in cultures of the humanembryonic stem cell line H7 (FIG. 23A) and the human embryonic stem cellline H9 (FIG. 23B) and the human embryonic stem cell line H1 (FIG. 23C).Results are expressed as fold increase over undifferentiated cells.Cells were treated according to the methods disclosed in Example 4.

FIGS. 24A and 24B depict phase contrast images of cultures of the humanembryonic stem cell line H9 at passage 46 in the presence of 100 ng/mlof activin A (FIG. 24A) or 100 ng/ml of activin A+20 ng/ml Wnt-3a (FIG.24B). Cells were treated for five days.

FIGS. 25A, 25B, 25C and 25D depict the expression of CXCR4 by FACS incultures of the human embryonic stem cell line H7 at passage 44 (FIGS.25A and 25B) and H9 at passage 46 (FIGS. 25C and 25D), followingtreatment according to the methods disclosed in Example 4. FIGS. 25B and25D show the effect of 20 ng/ml of Wnt-3a on CXCR4 expression. FIGS. 25Aand 25C show CXCR4 expression in the absence of Wnt-3a. Results wereobtained 5 days post treatment.

FIGS. 26A and 26B display the real-time PCR data for expression of thegenes indicated in cultures of the human embryonic stem cell line H7(FIG. 26A) and H9 (FIG. 26B). Cultures were treated with thedifferentiation protocol disclosed in Example 4. The effects of Wntagonists Wnt-3a (20 ng/ml), Wnt-5a (20 ng/ml) and Wnt-7a (20 ng/ml) werealso tested, as indicated in the panels. Cells were treated for 5 days.Results are expressed as fold increase over undifferentiated cells.

FIGS. 27A, 27B, 27C and 27D depict the expression of CXCR4 in culturesof the human embryonic stem cell line H9 at passage 46, by FACS at fivedays post treatment. FIG. 27A depicts CXCR4 expression in the absence ofWnt-3a. FIG. 27B depicts CXCR4 expression following treatment with 10ng/ml Wnt-3a. FIG. 27C depicts CXCR4 expression following treatment with20 ng/ml Wnt-3a, and FIG. 27D depicts CXCR4 expression followingtreatment with 50 ng/ml Wnt-3a.

FIGS. 28A, 28B and 28C depict the expression of definitive markersindicated in cultures of the human embryonic stem cell line H9 after 5days of treatment. Results are shown as fold increase in expression overuntreated cells, as determined by real-time PCR. FIG. 28A shows theeffect of 10, 20 and 50 ng/ml Wnt-3a on the expression of definitiveendoderm marker genes indicated. FIG. 28B shows the effect of 1, 5 or 10ng/ml Wnt-3a (x-axis labels: 10, 5, 1) on the expression on goosecoid(▪) and CXCR4 (□) expression, at 2 (2d) and 5 (5d) days post treatment.FIG. 28C shows the effect of 1, 5 or 10 ng/ml Wnt-3a on cell number, at2 days (□) or 5 days (▪).

FIGS. 29A, 29B, 29C, 29D, 29E, 29F, 29G and 29H depict the expression ofCXCR4 in cultures of the human embryonic stem cell line H9 by FACS,following a 5 day treatment with the differentiation protocol disclosedin Example 4. Cells were cultured in the absence of Wnt-3a or GSK-3Binhibitor (FIG. 29A), 20 ng/ml Wnt-3a for the entire 5 day period (FIG.29B), 1000 nM GSK-3B inhibitor IX for the entire 5 day period (FIG.29C), 500 nM GSK-3B inhibitor IX for the entire 5 day period (FIG. 29D),100 nM GSK-3B inhibitor IX for the entire 5 day period (FIG. 29E), 10 nMGSK-3B inhibitor IX for the entire 5 day period (FIG. 29F), 100 nMGSK-3B inhibitor IX for days 1-2 (FIG. 29G), 10 nM GSK-3B inhibitor IXfor days 1-2 (FIG. 29H).

FIGS. 30A and 30 B depict the gene expression of definitive endodermmarkers by real-time PCR. Results are expressed as fold increase overuntreated cells. FIG. 30A shows data obtained from the human embryoniccell line H9 at passage number 48, treated to the definitive endodermprotocol disclosed in Example 4, containing the Wnt-3a or GSK-3Binhibitor at the concentrations and the times indicated. FIG. 30B showsdata obtained from the human embryonic cell line H9 at passage number46, treated to the definitive endoderm protocol disclosed in Example 4,containing the Wnt-3a or GSK-3B inhibitor at the concentrations and thetimes indicated.

FIGS. 31A, 31B, 31C, 31D, 31E and 31F depict the expression of CXCR4 byFACS for embryonic stem cell lines used in the present invention. FIGS.31A, 31B, 31C and 31D show data obtained from the human embryonic stemcell line H9 at passage number 49. FIGS. 31E and 31F show data obtainedfrom the human embryonic stem cell line H1 at passage number 46. Datawas obtained 5 days post treatment. Cells were treated with thefollowing conditions: FIG. 31A: 10 ng/ml activin A for all five daysplus 20 ng/ml of Wnt-3a for the first two days; FIG. 31B: 100 ng/mlactivin A for all five days plus 20 ng/ml of Wnt-3a for the first twodays; FIG. 31C: 100 ng/ml activin A for all five days plus 100 nM ofGSK-3B inhibitor IX for the first two days; FIG. 31D: 10 ng/ml activin Afor all five days plus 100 nM GSK-3B IX inhibitor for the first twodays, FIG. 31E: 100 ng/ml activin A for all five days plus 20 ng/ml ofWnt-3a for the first two days, and FIG. 31F:10 ng/ml activin A for allfive days plus 20 ng/ml of Wnt-3a for the first two days.

FIGS. 32A and 32B depict the gene expression of definitive endodermmarkers, as determined by real-time PCR for cultures of the humanembryonic stem cell line H9 at passage 49, treated with 10, 50, or 100ng/ml of activin A plus 20 ng/ml of Wnt-3a: FIG. 32A: expression of AFP,Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and FIG. 32B: SOX-17 andGATA4. Results are expressed as fold increase over untreated cells.

FIGS. 33A, 33B, 33C, 33D, 33E and 33F depict the expression of CXCR4 byFACS for the embryonic stem cell line H9 at passage 53. Data wasobtained 5 days post treatment. Cells were treated with the followingconditions: FIG. 33A: 100 ng/ml activin A for all five days plus 20ng/ml of Wnt-3a for the first two days and 25 ng/ml BMP-4 for days 3-5;FIG. 33B: 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3afor the first two days; FIG. 33C: 100 ng/ml activin A for all five daysplus 100 nM of GSK-3B inhibitor IX for the first two days; FIG. 33D: 20ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days; FIG. 33E: 100 ng/mlactivin A for all five days plus 20 ng/ml of Wnt-3a+100 nm GSK-3Binhibitor IX for the first two days, and FIG. 33F: 100 ng/ml activinA+25 ng/ml BMP-4 for all five days. For all the panels, the X-axisrepresents expression of CD9 and the Y-axis represents expression ofCXCR4 (CD184).

FIGS. 34A and 34B depict the gene expression of definitive endodermmarkers, as determined by real-time PCR for cultures of the humanembryonic stem cell line H1 at passage 46, treated with 10 or 100 ng/mlof activin A plus ng/ml of Wnt-3a or 100 NM GSK-3B inhibitor: FIG. 34A:expression of AFP, Bry, CXCR4, GSC, and POU5F (Oct-4) and FIG. 34B:SOX-17, HNF-3B, and GATA4. Results are expressed as fold increase overuntreated cells.

FIGS. 35A and 35B depicts the gene expression of definitive endodermmarkers, as determined by real-time PCR for cultures of the humanembryonic stem cell line H9 at passage 49, treated with 50 or 100 ng/mlof activin A plus or 100 nM GSK-3B inhibitor: FIG. 35A: expression ofAFP, Bry, CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and FIG. 35B: SOX-17 andGATA4. Results are expressed as fold increase over untreated cells.

FIGS. 36A and 36B depicts the gene expression of definitive endodermmarkers, as determined by real-time PCR for cultures of the humanembryonic stem cell line H9 at passage 53, treated with combinations ofactivin A, Wnt-3a, GSK-3 inhibitor, and BMP-4, for five days: FIG. 36A:expression of AFP, Bry, CXCR4, GSC, HNF-3B, and SOX7 and FIG. 36B:SOX-17, HNF-3B and GATA4.

FIG. 37 depicts the percentage of CXCR4 expression, determined by FACS,in cultures of the human embryonic stem cell line H9, treated with theconditions listed in Example 22.

FIGS. 38A and 38B depict the expression of definitive endoderm markersas determined by FACS in cultures of the human embryonic stem cell lineH9, cultured on fibronectin (FIG. 38A) or MATRIGEL™ (FIG. 38B).

FIG. 39 depicts the expression of definitive endoderm markers asdetermined by real-time PCR in cultures of the human embryonic stem cellline H9, cultured on fibronectin (□) or a 1:10 dilution of growth factorreduced MATRIGEL (▪).

FIG. 40 depicts the effect of various concentrations of MATRIGEL in thepresence of low serum, 100 ng/ml of activin A and 20 ng/ml of Wnt-3a ondifferentiating human embryonic stem cells into definitive endoderm.Cells were treated according to the methods disclosed in Example 4.Results shown are the expression levels of the genes indicated, asdetermined by real-time PCR.

FIGS. 41A, 41B, 41C, 41D, 41E-1, 41E-2, 41F-1, 41F-2, 41G-1, 41G-2depicts the role of Wnt-3a in definitive endoderm formation by humanembryonic stem cells maintained on MATRIGEL, but differentiated on mouseembryonic fibroblasts. FIGS. 41A, 41B, 41C and 41D show real-time PCRdata for the genes indicated. FIGS. 41E-1, 41E-2, 41F-1, 41F-2, 41G-1,41G-2 show FACS data for the conditions indicated.

FIG. 42 shows the differentiation of human embryonic stem cells culturedon tissue culture substrate coated with MATRIGEL™ to definitive endodermfollowing treatment with the Wnt Inhibitor DKK-1. Results shown are theexpression of the genes indicated, as determined by real-time PCR in H9cells treated according to the methods disclosed in Example 4 in thepresence of 20 ng/ml of Wnt-3A plus 100 ng/ml of DKK1 (DE+DKK1), or inthe absence of DKK1 (DE).

FIGS. 43A and 43B show immunofluorescence staining of definitiveendoderm markers in cultures of the human embryonic stem cell line H9cultured on tissue culture substrate coated with MATRIGEL anddifferentiated in low serum plus 100 ng/ml of activin-A without (FIG.43A), or with (FIG. 43B) 20 ng/ml of Wnt-3a. Ecad=E-cadherin,NCAM=N-cadherin,

FIGS. 44A and 44B show the differentiation of the human embryonic stemcell line SA002 at passage 38 into definitive endoderm. Cells weretreated for five days with the conditions indicated and gene expressionwas determined by real-time PCR, for the genes indicated in FIGS. 44Aand 44B.

FIGS. 45A, 45B and 45C show the expression of CXCR4 by FACS in the humanembryonic stem cell line SA002 at passage 38, following treatment with100 ng/ml activin A treatment (FIG. 45A), 100 ng/ml activin A+20 ng/mlWnt-3a (FIG. 45B), or 100 ng/ml activin A+100 nM GSK-3B inhibitor IX(FIG. 45C). Cells were treated for five days.

FIGS. 46A and 46B show the differentiation of the human embryonic stemcell line H1 at passage 55 into definitive endoderm on tissue culturesubstrate coated with human serum. Cells were treated with theconditions indicated and gene expression was determined by real-timePCR, for the genes indicated in FIGS. 46A and 46B.

FIGS. 47A and 47B show the differentiation of cultures of the humanembryonic stem cell line H1 at P54, on tissue culture substrate coatedwith MATRIGEL™ to definitive endoderm. The effects of various GSK-Binhibitors were tested following a five-day DE protocol. The followingGSK-3B inhibitors were evaluated at 100 nM for the first two days oftreatment: GSK-3B VIII, IX, XI, and XII.

FIGS. 48A, 48B, 48C and 48D show the expression of AFP (FIG. 48A), Pdx-1(FIG. 48B), Cdx-2 and Glut-2 (FIG. 48C) and HNF-3beta, HNF-6 andsomatostatin (FIG. 48D) in cultures of the human embryonic stem cellline H9 at passage 49, cultured and treated according to the methodsdisclosed in Example 4 in the presence of 20 ng/ml of Wnt-3a for thefirst two days of treatment. Following the treatment, the cells weretreated for three additional days with 2% FBS plus 1 μM retinoic acid,0.1 to 1 μM TTNPB(4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid Arotinoid acid), or 0.1-10 μM AM-580(4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid). The cells were next treated for three additional days in 2% FBSplus 20 ng/ml of bFGF.

FIGS. 49A and 49B show the real-time PCR results of the expression ofthe definitive endoderm markers indicated in FIGS. 49A and 49B. incultures of the human embryonic stem cell line H1 treated with activin Aand Wnt-1 for the times and concentrations indicated.

DETAILED DESCRIPTION

For clarity of disclosure, and not by way of limitation, the detaileddescription of the invention is divided into subsections that describeor illustrate certain features, embodiments, or applications of thepresent invention.

Definitions

Stem cells are undifferentiated cells defined by their ability at thesingle cell level to both self-renew and differentiate to produceprogeny cells, including self-renewing progenitors, non-renewingprogenitors, and terminally differentiated cells. Stem cells are alsocharacterized by their ability to differentiate in vitro into functionalcells of various cell lineages from multiple germ layers (endoderm,mesoderm and ectoderm), as well as to give rise to tissues of multiplegerm layers following transplantation and to contribute substantially tomost, if not all, tissues following injection into blastocysts.

Stem cells are classified by their developmental potential as: (1)totipotent, meaning able to give rise to all embryonic andextraembryonic cell types; (2) pluripotent, meaning able to give rise toall embryonic cell types; (3) multipotent, meaning able to give rise toa subset of cell lineages, but all within a particular tissue, organ, orphysiological system (for example, hematopoietic stem cells (HSC) canproduce progeny that include HSC (self-renewal), blood cell restrictedoligopotent progenitors and all cell types and elements (e.g.,platelets) that are normal components of the blood); (4) oligopotent,meaning able to give rise to a more restricted subset of cell lineagesthan multipotent stem cells; and (5) unipotent, meaning able to giverise to a single cell lineage (e.g., spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”)or less specialized cell acquires the features of a specialized cellsuch as, for example, a nerve cell or a muscle cell. A differentiated ordifferentiation-induced cell is one that has taken on a more specialized(“committed”) position within the lineage of a cell. The term“committed”, when applied to the process of differentiation, refers to acell that has proceeded in the differentiation pathway to a point where,under normal circumstances, it will continue to differentiate into aspecific cell type or subset of cell types, and cannot, under normalcircumstances, differentiate into a different cell type or revert to aless differentiated cell type. De-differentiation refers to the processby which a cell reverts to a less specialized (or committed) positionwithin the lineage of a cell. As used herein, the lineage of a celldefines the heredity of the cell, i.e., which cells it came from andwhat cells it can give rise to. The lineage of a cell places the cellwithin a hereditary scheme of development and differentiation. Alineage-specific marker refers to a characteristic specificallyassociated with the phenotype of cells of a lineage of interest and canbe used to assess the differentiation of an uncommitted cell to thelineage of interest.

“AFP” or “alpha-fetoprotein protein” as used herein, refers to anantigen produced at the onset of liver development. AFP may also beexpressed in extraembryonic cells.

“Albumin” is a soluble monomeric protein that makes up about half of allserum proteins in adults.

“β-cell lineage” refer to cells with positive gene expression for thetranscription factor PDX-1 and at least one of the followingtranscription factors: NGN-3, Nkx2.2, Nkx6.1, NeuroD, Isl-1, HNF-3 beta,MAFA, Pax4, and Pax6. Cells expressing markers characteristic of the βcell lineage include β cells.

“Brachyury”, as used herein, is a T-box gene family member. It is themarker for primitive streak and mesoderm cells.

“Cells expressing markers characteristic of the definitive endodermlineage” as used herein refer to cells expressing at least one of thefollowing markers: SOX-17, GATA-4, HNF-3 beta, GSC, Cer1, Nodal, FGF8,Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES),DKK4, FGF17, GATA-6, CXCR4, C-Kit, CD99, or OTX2. Cells expressingmarkers characteristic of the definitive endoderm lineage includeprimitive streak precursor cells, primitive streak cells, mesendodermcells and definitive endoderm cells.

“c-Kit” and “CD117” both refer to a cell surface receptor tyrosinekinase having a sequence disclosed in Genbank Accession No. X06182, or anaturally occurring variant sequence thereof (e.g., allelic variant).

“CD99” as used herein refers to the protein encoded by the gene with theaccession number NM 002414.

“Cells expressing markers characteristic of the pancreatic endodermlineage” as used herein refer to cells expressing at least one of thefollowing markers: PDX-1, HNF-1beta, PTF-1 alpha, HNF-6, or HB9. Cellsexpressing markers characteristic of the pancreatic endoderm lineageinclude pancreatic endoderm cells.

“Cells expressing markers characteristic of the pancreatic endocrinelineage” as used herein refer to cells expressing at least one of thefollowing markers: NGN-3, NeuroD, Islet-1, PDX-1, NKX6.1, Pax-4, Ngn-3,or PTF-1 alpha. Cells expressing markers characteristic of thepancreatic endocrine lineage include pancreatic endocrine cells,pancreatic hormone expressing cells, and pancreatic hormone secretingcells, and cells of the β-cell lineage.

“Cer1” or “Cerebrus” as used herein is a member of the cysteine knotsuperfamily of proteins.

“CXCR4” as used herein refers to the stromal cell-derived factor 1(SDF-1) receptor, also known as “LESTR” or “fusin”. In the gastrulatingmouse embryo, CXCR4 is expressed in the definitive endoderm and mesodermbut not in extraembryonic endoderm.

“Definitive endoderm” as used herein refers to cells which bear thecharacteristics of cells arising from the epiblast during gastrulationand which form the gastrointestinal tract and its derivatives.Definitive endoderm cells express the following markers: HNF-3 beta,GATA-4, SOX-17, Cerberus, OTX2, goosecoid, C-Kit, CD99, and Mixl1.

“Extraembryonic endoderm” as used herein refers to a population of cellsexpressing at least one of the following markers: SOX-7, AFP, and SPARC.

“FGF-2”, “FGF-4” “FGF-8” “FGF-10”, and “FGF-17” as used herein, aremembers of the fibroblast growth factor family.

“GATA-4” and “GATA-6” are members of the GATA transcription factorfamily. This family of transcription factors is induced by TGF-βsignaling, and contribute to the maintenance of early endoderm markers.

“GLUT-2”, as used herein, refers to the glucose transporter moleculethat is expressed in numerous fetal and adult tissues, includingpancreas, liver, intestine, brain, and kidney.

“Goosecoid” or “GSC” as used herein, refers to a homeodomaintranscription factor expressed in the dorsal lip of the blastopore.

“HB9” as used herein, refers to the homeobox gene 9.

“HNF-1 alpha”, “HNF-1 beta”, “HNF-3 beta”, and “HNF-6” belong to thehepatic nuclear factor family of transcription factors, which ischaracterized by a highly conserved DNA binding domain and two shortcarboxy-terminal domains.

“Islet-1” or “Isl-1” as used herein is a member of the LIM/homeodomainfamily of transcription factors, and is expressed in the developingpancreas.

“MafA” as used herein is a transcription factor expressed in thepancreas, and controls the expression of genes involved in insulinbiosynthesis and secretion.

“Markers” as used herein, are nucleic acid or polypeptide molecules thatare differentially expressed in a cell of interest. In this context,differential expression means an increased level for a positive markerand a decreased level for a negative marker. The detectable level of themarker nucleic acid or polypeptide is sufficiently higher or lower inthe cells of interest compared to other cells, such that the cell ofinterest can be identified and distinguished from other cells using anyof a variety of methods known in the art.

“Mesendoderm cell” as used herein refers to a cell expressing at leastone of the following markers: CD48, eomesodermin (EOMES), SOX-17, DKK4,HNF-3 beta, GSC, FGF17, GATA-6.

“Mixl1” as used herein refers to a homeobox gene, which is marker forthe cells in the primitive streak, mesoderm, and endoderm.

“NeuroD” as used herein is basic helix-loop-helix (bHLH) transcriptionfactor implicated in neurogenesis.

“NGN-3” as used herein, is a member of the neurogenin family of basicloop-helix-loop transcription factors.

“Nkx-2.2” and “Nkx-6.1” as used herein are members of the Nkxtranscription factor family.

“Nodal” as used herein, is a member of the TGF beta superfamily ofproteins.

“Oct-4” is a member of the POU-domain transcription factor and is widelyregarded as a hallmark of pluripotent stem cells. The relationship ofOct-4 to pluripotent stem cells is indicated by its tightly restrictedexpression to undifferentiated pluripotent stem cells. Upondifferentiation to somatic lineages, the expression of Oct-4 disappearsrapidly.

“Pancreatic endocrine cell”, or “pancreatic hormone expressing cell” asused herein refers to a cell capable of expressing at least one of thefollowing hormones: insulin, glucagon, somatostatin, and pancreaticpolypeptide.

“Pancreatic hormone secreting cell” as used herein refers to a cellcapable of secreting at least one of the following hormones: insulin,glucagon, somatostatin, and pancreatic polypeptide.

“Pax-4” and “Pax-6” as used herein are pancreatic p cell specifictranscription factors that are implicated in islet development.

“PDX-1” as used herein refers to a homeodomain transcription factorimplicated in pancreas development.

“Pre-primitive streak cell” as used herein refers to a cell expressingat least one of the following markers: Nodal, or FGF8

“Primitive streak cell” as used herein refers to a cell expressing atleast one of the following markers: Brachyury, Mix-like homeoboxprotein, or FGF4.

“PTF-1 alpha” as used herein refers to a basic helix-loop-helix proteinof 48 kD that is a sequence-specific DNA-binding subunit of the trimericpancreas transcription factor-1 (PTF1).

“SOX 1”, “SOX-2”, “SOX 7”, and “SOX-17” as used herein, are a members ofthe SOX transcription factor family, and are implicated inembryogenesis.

“SPARC” as used herein is also known as “secreted protein acidic andrich in cysteine”.

“SSEA-1” (Stage Specific Embryonic Antigen-1) is a glycolipid surfaceantigen present on the surface of murine teratocarcinoma stem cells(EC), murine and human embryonic germ cells (EG), and murine embryonicstem cells (ES).

“SSEA-3” (Stage Specific Embryonic Antigen-3) is a glycolipid surfaceantigen present on the surface of human teratocarcinoma stem cells (EC),human embryonic germ cells (EG), and human embryonic stem cells (ES).

“SSEA-4” (Stage Specific Embryonic Antigen-4) is a glycolipid surfaceantigen present on the surface of human teratocarcinoma stem cells (EC),human embryonic germ cells (EG), and human embryonic stem cells (ES).

“TRA1-60” is a keratin sulfate related antigen that is expressed on thesurface of human teratocarcinoma stem cells (EC), human embryonic germcells (EG), and human embryonic stem cells (ES).

“TRA1-81” is a keratin sulfate related antigen that is expressed on thesurface of human teratocarcinoma stem cells (EC), human embryonic germcells (EG), and human embryonic stem cells (ES).

“TRA2-49” is an alkaline phosphatase isozyme expressed on the surface ofhuman teratocarcinoma stem cells (EC) and human embryonic stem cells(ES).

“ITF-1” as used herein, refers a transcriptional coactivator expressedin pluripotent embryonic stem cells and extra-embryonic cells.

“Zic1” as used herein is a member of the Zic transcription factorfamily, Zic1 regulates the expression of neural and neuralcrest-specific genes and is expressed in the cells of the dorsal neuraltube and the premigratory neural crest.

Isolation, Expansion and Culture of Pluripotent Stem CellsCharacterization of Pluripotent Stem Cells

Pluripotent stem cells may express one or more of the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Differentiation of pluripotent stem cells in vitroresults in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (ifpresent) and increased expression of SSEA-1. Undifferentiatedpluripotent stem cells typically have alkaline phosphatase activity,which can be detected by fixing the cells with 4% paraformaldehyde, andthen developing with Vector Red as a substrate, as described by themanufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiatedpluripotent stem cells also typically express Oct-4 and TERT, asdetected by RT-PCR.

Another desirable phenotype of propagated pluripotent stem cells is apotential to differentiate into cells of all three germinal layers:endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotentstem cells can be confirmed, for example, by injecting cells into severecombined immunodeficient (SCID) mice, fixing the teratomas that formusing 4% paraformaldehyde, and then examining them histologically forevidence of cell types from the three germ layers. Alternatively,pluripotency may be determined by the creation of embryoid bodies andassessing the embryoid bodies for the presence of markers associatedwith the three germinal layers.

Propagated pluripotent stem cell lines may be karyotyped using astandard G-banding technique and compared to published karyotypes of thecorresponding primate species. It is desirable to obtain cells that havea “normal karyotype,” which means that the cells are euploid, whereinall human chromosomes are present and not noticeably altered.

Sources of Pluripotent Stem Cells

The types of pluripotent stem cells that may be used include establishedlines of pluripotent cells derived from tissue formed after gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily before approximately 10-12 weeksgestation. Non-limiting examples are established lines of humanembryonic stem cells or human embryonic germ cells, such as, for examplethe human embryonic stem cell lines H1, H7, and H9 (WiCell). Alsocontemplated is use of the compositions of this disclosure during theinitial establishment or stabilization of such cells, in which case thesource cells would be primary pluripotent cells taken directly from thesource tissues. Also suitable are cells taken from a pluripotent stemcell population already cultured in the absence of feeder cells. Alsosuitable are mutant human embryonic stem cell lines, such as, forexample, BG01v (BresaGen, Athens, GA).

In one embodiment, human embryonic stem cells are prepared as describedby Thomson et al. (U.S. Pat. No. 5,843,780; Science 282:1145, 1998;Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A.92:7844, 1995).

Culture of Pluripotent Stem Cells

In one embodiment, pluripotent stem cells are typically cultured on alayer of feeder cells that support the pluripotent stem cells in variousways. Alternatively, pluripotent stem cells are cultured in a culturesystem that is essentially free of feeder cells, but nonethelesssupports proliferation of pluripotent stem cells without undergoingsubstantial differentiation. The growth of pluripotent stem cells infeeder-free culture without differentiation is supported using a mediumconditioned by culturing previously with another cell type.Alternatively, the growth of pluripotent stem cells in feeder-freeculture without differentiation is supported using a chemically definedmedium.

For example, Reubinoff et al (Nature Biotechnology 18: 399-404 (2000))and Thompson et al (Science 6 Nov. 1998: Vol. 282. no. 5391, pp.1145-1147) disclose the culture of pluripotent stem cell lines fromhuman blastocysts using a mouse embryonic fibroblast feeder cell layer.

Richards et al, (Stem Cells 21: 546-556, 2003) evaluated a panel of 11different human adult, fetal and neonatal feeder cell layers for theirability to support human pluripotent stem cell culture. Richards et al,states: “human embryonic stem cell lines cultured on adult skinfibroblast feeders retain human embryonic stem cell morphology andremain pluripotent”.

US20020072117 discloses cell lines that produce media that support thegrowth of primate pluripotent stem cells in feeder-free culture. Thecell lines employed are mesenchymal and fibroblast-like cell linesobtained from embryonic tissue or differentiated from embryonic stemcells.

US20020072117 also discloses the use of the cell lines as a primaryfeeder cell layer.

In another example, Wang et al (Stem Cells 23: 1221-1227, 2005)discloses methods for the long-term growth of human pluripotent stemcells on feeder cell layers derived from human embryonic stem cells.

In another example, Stojkovic et al (Stem Cells 2005 23: 306-314, 2005)disclose a feeder cell system derived from the spontaneousdifferentiation of human embryonic stem cells.

In a further example, Miyamoto et al (Stem Cells 22: 433-440, 2004)disclose a source of feeder cells obtained from human placenta.

Amit et al (Biol. Reprod 68: 2150-2156, 2003) discloses a feeder celllayer derived from human foreskin.

In another example, Inzunza et al (Stem Cells 23: 544-549, 2005)disclose a feeder cell layer from human postnatal foreskin fibroblasts.

U.S. Pat. No. 6,642,048 discloses media that support the growth ofprimate pluripotent stem (pPS) cells in feeder-free culture, and celllines useful for production of such media. U.S. Pat. No. 6,642,048states: “This invention includes mesenchymal and fibroblast-like celllines obtained from embryonic tissue or differentiated from embryonicstem cells. Methods for deriving such cell lines, processing media, andgrowing stem cells using the conditioned media are described andillustrated in this disclosure.”

In another example, WO2005014799 discloses conditioned medium for themaintenance, proliferation and differentiation of mammalian cells.WO2005014799 states: “The culture medium produced in accordance with thepresent invention is conditioned by the cell secretion activity ofmurine cells, in particular, those differentiated and immortalizedtransgenic hepatocytes, named MMH (Met Murine Hepatocyte).”

In another example, Xu et al (Stem Cells 22: 972-980, 2004) disclosesconditioned medium obtained from human embryonic stem cell derivativesthat have been genetically modified to over express human telomerasereverse transcriptase.

In another example, US20070010011 discloses a chemically defined culturemedium for the maintenance of pluripotent stem cells.

An alternative culture system employs serum-free medium supplementedwith growth factors capable of promoting the proliferation of embryonicstem cells. For example, Cheon et al (BioReprodDOI:10.1095/biolreprod.105.046870, Oct. 19, 2005) disclose afeeder-free, serum-free culture system in which embryonic stem cells aremaintained in unconditioned serum replacement (SR) medium supplementedwith different growth factors capable of triggering embryonic stem cellself-renewal.

In another example, Levenstein et al (Stem Cells 24: 568-574, 2006)disclose methods for the long-term culture of human embryonic stem cellsin the absence of fibroblasts or conditioned medium, using mediasupplemented with bFGF.

In another example, US20050148070 discloses a method of culturing humanembryonic stem cells in defined media without serum and withoutfibroblast feeder cells, the method comprising: culturing the stem cellsin a culture medium containing albumin, amino acids, vitamins, minerals,at least one transferrin or transferrin substitute, at least one insulinor insulin substitute, the culture medium essentially free of mammalianfetal serum and containing at least about 100 ng/ml of a fibroblastgrowth factor capable of activating a fibroblast growth factor signalingreceptor, wherein the growth factor is supplied from a source other thanjust a fibroblast feeder layer, the medium supported the proliferationof stem cells in an undifferentiated state without feeder cells orconditioned medium.

In another example, US20050233446 discloses a defined media useful inculturing stem cells, including undifferentiated primate primordial stemcells. In solution, the media is substantially isotonic as compared tothe stem cells being cultured. In a given culture, the particular mediumcomprises a base medium and an amount of each of bFGF, insulin, andascorbic acid necessary to support substantially undifferentiated growthof the primordial stem cells.

In another example, U.S. Pat. No. 6,800,480 states “In one embodiment, acell culture medium for growing primate-derived primordial stem cells ina substantially undifferentiated state is provided which includes a lowosmotic pressure, low endotoxin basic medium that is effective tosupport the growth of primate-derived primordial stem cells. The basicmedium is combined with a nutrient serum effective to support the growthof primate-derived primordial stem cells and a substrate selected fromthe group consisting of feeder cells and an extracellular matrixcomponent derived from feeder cells. The medium further includesnon-essential amino acids, an anti-oxidant, and a first growth factorselected from the group consisting of nucleosides and a pyruvate salt.”

In another example, US20050244962 states: “In one aspect the inventionprovides a method of culturing primate embryonic stem cells. Onecultures the stem cells in a culture essentially free of mammalian fetalserum (preferably also essentially free of any animal serum) and in thepresence of fibroblast growth factor that is supplied from a sourceother than just a fibroblast feeder layer. In a preferred form, thefibroblast feeder layer, previously required to sustain a stem cellculture, is rendered unnecessary by the addition of sufficientfibroblast growth factor.”

In a further example, WO2005065354 discloses a defined, isotonic culturemedium that is essentially feeder-free and serum-free, comprising: a. abasal medium; b. an amount of bFGF sufficient to support growth ofsubstantially undifferentiated mammalian stem cells; c. an amount ofinsulin sufficient to support growth of substantially undifferentiatedmammalian stem cells; and d. an amount of ascorbic acid sufficient tosupport growth of substantially undifferentiated mammalian stem cells.

In another example, WO2005086845 discloses a method for maintenance ofan undifferentiated stem cell, said method comprising exposing a stemcell to a member of the transforming growth factor-beta (TGFβ) family ofproteins, a member of the fibroblast growth factor (FGF) family ofproteins, or nicotinamide (NIC) in an amount sufficient to maintain thecell in an undifferentiated state for a sufficient amount of time toachieve a desired result.

The pluripotent stem cells may be plated onto a suitable culturesubstrate. In one embodiment, the suitable culture substrate is anextracellular matrix component, such as, for example, those derived frombasement membrane or that may form part of adhesion moleculereceptor-ligand couplings. In one embodiment, a the suitable culturesubstrate is Matrigel® (Becton Dickenson). Matrigel® is a solublepreparation from Engelbreth-Holm-Swarm tumor cells that gels at roomtemperature to form a reconstituted basement membrane.

Other extracellular matrix components and component mixtures aresuitable as an alternative. Depending on the cell type beingproliferated, this may include laminin, fibronectin, proteoglycan,entactin, heparan sulfate, and the like, alone or in variouscombinations.

The pluripotent stem cells may be plated onto the substrate in asuitable distribution and in the presence of a medium that promotes cellsurvival, propagation, and retention of the desirable characteristics.All these characteristics benefit from careful attention to the seedingdistribution and can readily be determined by one of skill in the art.

Suitable culture media may be made from the following components, suchas, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco#11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco#10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco#15039-027; non-essential amino acid solution, Gibco 11140-050;β-mercaptoethanol, Sigma #M7522; human recombinant basic fibroblastgrowth factor (bFGF), Gibco #13256-029.

Differentiation of Pluripotent Stem Cells into Cells Expressing MarkersCharacteristic of the Pancreatic Endocrine Lineage

Pluripotent stem cells suitable for use in the present inventioninclude, for example, the human embryonic stem cell line H9 (NIH code:WA09), the human embryonic stem cell line H1 (NIH code: WA01), the humanembryonic stem cell line H7 (NIH code: WA07), and the human embryonicstem cell line SA002 (Cellartis, Sweden). Also suitable for use in thepresent invention are cells that express at least one of the followingmarkers characteristic of pluripotent cells: ABCG2, cripto, CD9, FoxD3,Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT, UTF-1, ZFP42, SSEA-3,SSEA-4, Tra1-60, Tra1-81.

Markers characteristic of the definitive endoderm lineage are selectedfrom the group consisting of SOX17, GATA4, Hnf-3beta, GSC, Cer1, Nodal,FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin(EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable foruse in the present invention is a cell that expresses at least one ofthe markers characteristic of the definitive endoderm lineage. In oneaspect of the present invention, a cell expressing markerscharacteristic of the definitive endoderm lineage is a primitive streakprecursor cell. In an alternate aspect, a cell expressing markerscharacteristic of the definitive endoderm lineage is a mesendoderm cell.In an alternate aspect, a cell expressing markers characteristic of thedefinitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selectedfrom the group consisting of Pdx1, HNF-1beta, PTF1a, HNF-6, 11B9 andPROX1. Suitable for use in the present invention is a cell thatexpresses at least one of the markers characteristic of the pancreaticendoderm lineage. In one aspect of the present invention, a cellexpressing markers characteristic of the pancreatic endoderm lineage isa pancreatic endoderm cell.

Markers characteristic of the pancreatic endocrine lineage are selectedfrom the group consisting of NGN-3, NeuroD, Islet-1, Pdx-1, NKX6.1,Pax-4, Ngn-3, and PTF-1 alpha. In one embodiment, a pancreatic endocrinecell is capable of expressing at least one of the following hormones:insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitablefor use in the present invention is a cell that expresses at least oneof the markers characteristic of the pancreatic endocrine lineage. Inone aspect of the present invention, a cell expressing markerscharacteristic of the pancreatic endocrine lineage is a pancreaticendocrine cell. The pancreatic endocrine cell may be a pancreatichormone expressing cell. Alternatively, the pancreatic endocrine cellmay be a pancreatic hormone secreting cell.

In one aspect of the present invention, the pancreatic endocrine cell isa cell expressing markers characteristic of the β cell lineage. A cellexpressing markers characteristic of the β cell lineage expresses Pdx1and at least one of the following transcription factors: NGN-3, Nkx2.2,Nkx6.1, NeuroD, Isl-1, HNF-3 beta, MAFA, Pax4, and Pax6. In one aspectof the present invention, a cell expressing markers characteristic ofthe β cell lineage is a β cell.

Formation of Cells Expressing Markers Characteristic of the DefinitiveEndoderm Lineage

Pluripotent stem cells may be differentiated into cells expressingmarkers characteristic of the definitive endoderm lineage by any methodin the art or by any method proposed in this invention.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al, NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in Shinozaki et al, Development 131,1651-1662 (2004).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in McLean et al, Stem Cells 25, 29-38(2007).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineageaccording to the methods disclosed in D'Amour et al, NatureBiotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A and serum,and then culturing the cells with activin A and serum of a differentconcentration. An example of this method is disclosed in NatureBiotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A inthe absence of serum, then culturing the cells with activin A with serumof another concentration. An example of this method is disclosed inD'Amour et al, Nature Biotechnology, 2005.

For example, pluripotent stem cells may be differentiated into cellsexpressing markers characteristic of the definitive endoderm lineage byculturing the pluripotent stem cells in medium containing activin A anda Wnt ligand in the absence of serum, then removing the Wnt ligand andculturing the cells with activin A with serum. An example of this methodis disclosed in Nature Biotechnology 24, 1392-1401 (2006).

In one aspect of the present invention, pluripotent stem cells may bedifferentiated into cells expressing markers characteristic of thedefinitive endoderm lineage by plating the pluripotent stem cells on atissue culture substrate coated with an extracellular matrix, thenculturing the pluripotent stem cells with activin A and a Wnt ligand ina first culture medium containing serum for a period of time, and thenculturing the pluripotent stem cells with activin A in a second culturemedium containing a greater concentration of serum for about anotherperiod of time.

The concentration of serum in the first culture medium disclosed abovemay be from about zero to about 0.5 percent, and the culture time may befrom about one to about three days. The concentration of serum in thesecond culture medium disclosed above may be from about 0.5 percent toabout two percent, and the culture time may be from about one to aboutfour days.

In an alternate embodiment of the present invention, pluripotent stemcells may be differentiated into cells expressing markers characteristicof the definitive endoderm lineage by plating the pluripotent stem cellson a tissue culture substrate coated with an extracellular matrix, thenculturing the pluripotent stem cells with activin A and a Wnt ligand ina first culture medium containing serum for about a period of time, andthen culturing the pluripotent stem cells with activin A and a Wntligand in a second culture medium containing a greater concentration ofserum for another period of time.

The concentration of serum in the first culture medium disclosed abovemay be from about zero to about 0.5 percent, and the culture time may befrom about one to about three days. The concentration of serum in thesecond culture medium disclosed above may be from about 0.5 percent toabout two percent, and the culture time may be from about one to aboutfour days.

In one embodiment, the present invention provides a method fordifferentiating pluripotent stem cells expressing markers characteristicof the definitive endoderm lineage, comprising the steps of:

-   -   a. Plating the pluripotent stem cells on a tissue culture        substrate coated with an extracellular matrix, and    -   b. Culturing the pluripotent stem cells with activin A and a Wnt        ligand.

Culturing the pluripotent stem cells with activin A and a Wnt ligand maybe performed in a single culture medium. Alternatively, culturing thepluripotent stem cells with activin A and a Wnt ligand may be performedseparately or together in more than one culture media. In oneembodiment, culturing the pluripotent stem cells with activin A and aWnt ligand is performed in two culture media.

Extracellular Matrix

In one aspect of the present invention, the pluripotent stem cells arecultured and differentiated on a tissue culture substrate coated with anextracellular matrix. The extracellular matrix may be a solubilizedbasement membrane preparation extracted from mouse sarcoma cells (whichis sold by BD Biosciences under the trade name MATRIGEL). Alternatively,the extracellular matrix may be growth factor-reduced MATRIGEL.

Alternatively, the extracellular matrix may fibronectin. In an alternateembodiment, the pluripotent stem cells are cultured and differentiatedon tissue culture substrate coated with human serum.

The extracellular matrix may be diluted prior to coating the tissueculture substrate. Examples of suitable methods for diluting theextracellular matrix and for coating the tissue culture substrate may befound in Kleinman, H. K., et al., Biochemistry 25:312 (1986), andHadley, M. A., et al., J. Cell. Biol. 101:1511 (1985).

In one embodiment, the extracellular matrix is MATRIGEL. In oneembodiment, the tissue culture substrate is coated with MATRIGEL at a1:10 dilution. In an alternate embodiment, the tissue culture substrateis coated with MATRIGEL at a 1:15 dilution. In an alternate embodiment,the tissue culture substrate is coated with MATRIGEL at a 1:30 dilution.In an alternate embodiment, the tissue culture substrate is coated withMATRIGEL at a 1:60 dilution.

In one embodiment, the extracellular matrix is growth factor-reducedMATRIGEL. In one embodiment, the tissue culture substrate is coated withgrowth factor-reduced MATRIGEL at a 1:10 dilution. In an alternateembodiment, the tissue culture substrate is coated with growthfactor-reduced MATRIGEL at a 1:15 dilution. In an alternate embodiment,the tissue culture substrate is coated with growth factor-reducedMATRIGEL at a 1:30 dilution. In an alternate embodiment, the tissueculture substrate is coated with growth factor-reduced MATRIGEL at a1:60 dilution.

Differentiation of Pluripotent Stem Cells into Cells Expressing MarkersCharacteristic of the Definitive Endoderm Lineage on an ExtracellularMatrix, Using a Single Culture Medium

When a single culture medium is used, it should contain sufficiently lowconcentrations of certain factors to allow the differentiation ofpluripotent stem cells to definitive endoderm, such as, for exampleinsulin and IGF (as disclosed in WO2006020919). This may be achieved bylowing the serum concentration, or alternatively, by using chemicallydefined media that lacks insulin and IGF. Examples of chemically definedmedia are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).

The culture medium may have a serum concentration in the range of about0% to about 10%. In an alternate embodiment, the concentration may be inthe range of about 0% to about 5%. In an alternate embodiment, theconcentration may be in the range of about 0% to about 2%. In analternate embodiment, the concentration may be about 2%.

The time of culturing with activin A and a Wnt ligand may range fromabout 1 day to about 7 days. In an alternate embodiment, the time ofculturing may range from about 1 day to about 3 days. In an alternateembodiment, the time of culturing may be about 3 days.

Activin A may be used at any concentration suitable to causedifferentiation of the pluripotent stem cells. The concentration maybefrom about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, theconcentration may be about 1 pg/ml to about 1 μg/ml. In anotheralternate embodiment, the concentration may be about 1 pg/ml to about100 ng/ml. In another alternate embodiment, the concentration may beabout 50 ng/ml to about 100 ng/ml. In another alternate embodiment, theconcentration may be about 100 ng/ml.

The choice of the Wnt ligand may be optimized to improve the efficiencyof the differentiation process. The Wnt ligand may be selected from thegroup consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment,the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand isWnt-3a.

The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000ng/ml. In an alternate embodiment, the concentration may be about 10ng/ml to about 100 ng/ml.

The single culture medium may also contain a GSK-3B inhibitor. TheGSK-3B inhibitor may be selected from the group consisting of GSK-3Binhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3Binhibitor is GSK-3B inhibitor IX. I did not see the definitions of IXand XI inhibitors. I did have these in the original WNT patentapplication.

When culturing pluripotent stem cells with a GSK-3B inhibitor, theconcentration of the GSK-3B inhibitor may be from about 1 nM to about1000 nM. In an alternate embodiment, the pluripotent stem cells arecultured with the GSK-3B inhibitor at a concentration of about 10 nM toabout 100 nM.

The single culture medium may also contain at least one other additionalfactor that may enhance the formation of cells expressing markerscharacteristic of the definitive endoderm lineage from pluripotent stemcells. Alternatively, the at least one other additional factor mayenhance the proliferation of the ells expressing markers characteristicof the definitive endoderm lineage formed by the methods of the presentinvention. Further, the at least one other additional factor may enhancethe ability of the ells expressing markers characteristic of thedefinitive endoderm lineage formed by the methods of the presentinvention to form other cell types, or improve the efficiency of anyother additional differentiation steps.

The at least one additional factor may be, for example, nicotinamide,members of the TGF-β family, including TGF-β1, 2, and 3, serum albumin,members of the fibroblast growth factor family, platelet-derived growthfactor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I,II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagonlike peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody,Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone,aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermalgrowth factor (EGF), gastrin I and II, copper chelators such as, forexample, triethylene pentamine, forskolin, Na-Butyrate, activin,betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid,trichostatin A, sodium butyrate, hepatocyte growth factor (HGF),sphingosine 1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco,CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA),keratinocyte growth factor (KGF), Dickkopf protein family, bovinepituitary extract, islet neogenesis-associated protein (INGAP), Indianhedgehog, sonic hedgehog, proteasome inhibitors, notch pathwayinhibitors, sonic hedgehog inhibitors, or combinations thereof.

The at least one other additional factor may be supplied by conditionedmedia obtained from pancreatic cells lines such as, for example, PANC-1(ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No:CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, forexample, HepG2 (ATCC No: HTB-8065), intestinal cell lines such as, forexample, FHs 74 (ATCC No: CCL-241), and primary or transformedendothelial cells.

Differentiation of Pluripotent Stem Cells into Cells Expressing MarkersCharacteristic of the Definitive Endoderm Lineage on an ExtracellularMatrix, Using Two Culture Media

Differentiation of pluripotent stem cells into cells of a definitiveendoderm lineage may be accomplished by culturing the pluripotent stemcells with activin A and a Wnt ligand using two culture media. Thus, thedifferentiation of the pluripotent stem cells may be accomplished asfollows:

-   -   a. Plating the pluripotent stem cells on a tissue culture        substrate coated with an extracellular matrix,    -   b. Culturing the pluripotent stem cells with activin A and a Wnt        ligand in a first culture medium, and    -   c. Culturing the pluripotent stem cells with activin A in a        second culture medium.

The first culture medium may contain serum at a low concentration, andthe second culture medium may contain serum at a higher concentrationthan the first culture medium.

The second culture medium may contain a Wnt ligand.

First Culture Medium: The first culture medium should containsufficiently low concentrations of certain factors to allow thedifferentiation of pluripotent stem cells into cells expressing markerscharacteristic of the definitive endoderm lineage, such as, for exampleinsulin and IGF (as disclosed in WO2006020919). This may be achieved bylowing the serum concentration, or alternatively, by using chemicallydefined media that lacks insulin and IGF. Examples of chemically definedmedia are disclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25;247(1):241-8.).

In the first culture medium there may be a lower concentration of serum,relative to the second culture medium. Increasing the serumconcentration in the second culture medium increases the survival of thecells, or, alternatively, may enhance the proliferation of the cells.The serum concentration of the first medium may be in the range of about0% to about 10%. Alternatively, the serum concentration of the firstmedium may be in the range of about 0% to about 2%. Alternatively, theserum concentration of the first medium may be in the range of about 0%to about 1%. Alternatively, the serum concentration of the first mediummay be about 0.5%.

When culturing the pluripotent stem cells with activin A and a Wntligand using at least two culture media, the time of culturing in thefirst culture medium may range from about 1 day to about 3 days.

Activin A may be used at any concentration suitable to causedifferentiation of the pluripotent stem cells. The concentration maybefrom about 1 pg/ml to about 100 μg/ml. In an alternate embodiment, theconcentration may be about 1 pg/ml to about 1 μg/ml. In anotheralternate embodiment, the concentration may be about 1 pg/ml to about100 ng/ml. In another alternate embodiment, the concentration may beabout 50 ng/ml to about 100 ng/ml. In another alternate embodiment, theconcentration may be about 100 ng/ml.

The choice of the Wnt ligand may be optimized to improve the efficiencyof the differentiation process. The Wnt ligand may be selected from thegroup consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment,the Wnt ligand is Wnt-1. In an alternate embodiment, the Wnt ligand isWnt-3a.

The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000ng/ml. In an alternate embodiment, the concentration may be about 10ng/ml to about 100 ng/ml.

The first culture medium may also contain a GSK-3B inhibitor. The GSK-3Binhibitor may be added to the first culture medium, to the secondculture medium, or to both the first and second culture media.

The GSK-3B inhibitor may be selected from the group consisting of GSK-3Binhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3Binhibitor is GSK-3B inhibitor IX.

When culturing pluripotent stem cells with a GSK-3B inhibitor, theconcentration of the GSK-3B inhibitor may be from about 1 nM to about1000 nM. In an alternate embodiment, the pluripotent stem cells arecultured with the GSK-3B inhibitor at a concentration of about 10 nM toabout 100 nM.

The first culture medium may also contain at least one other additionalfactor that may enhance the formation of cells expressing markerscharacteristic of the definitive endoderm lineage from pluripotent stemcells. Alternatively, the at least one other additional factor mayenhance the proliferation of the cells expressing markers characteristicof the definitive endoderm lineage formed by the methods of the presentinvention. Further, the at least one other additional factor may enhancethe ability of the cells expressing markers characteristic of thedefinitive endoderm lineage formed by the methods of the presentinvention to form other cell types, or improve the efficiency of anyother additional differentiation steps.

The at least one additional factor may be, for example, nicotinamide,members of TGF-β family, including TGF-β1, 2, and 3, serum albumin,members of the fibroblast growth factor family, platelet-derived growthfactor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I,II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagonlike peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody,Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone,aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermalgrowth factor (EGF), gastrin I and II, copper chelators such as, forexample, triethylene pentamine, forskolin, Na-Butyrate, activin,betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid,trichostatin A, sodium butyrate, hepatocyte growth factor (HGF),sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco,CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA),keratinocyte growth factor (KGF), Dickkopf protein family, bovinepituitary extract, islet neogenesis-associated protein (INGAP), Indianhedgehog, sonic hedgehog, proteasome inhibitors, notch pathwayinhibitors, sonic hedgehog inhibitors, or combinations thereof.

The at least one other additional factor may be supplied by conditionedmedia obtained from pancreatic cells lines such as, for example, PANC-1(ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No:CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, forexample, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as,for example, FHs 74 (ATCC No: CCL-241).

Second Culture Medium: The second culture medium should contain certainfactors, such as, for example, insulin and IGF (as disclosed inWO2006020919), at a sufficient concentration to promote the survival ofthe cultured cells. This may be achieved by increasing the serumconcentration, or, alternatively, by using chemically defined mediawhere the concentrations of insulin and IGF are increased relative tothe first culture medium. Examples of chemically defined media aredisclosed in Wiles et al (Exp Cell Res. 1999 Feb. 25; 247(1):241-8.).

In a second culture medium having higher concentrations of serum, theserum concentration of the second culture medium may be in the rangeabout 0.5% to about 10%. Alternatively, the serum concentration of thesecond culture medium may be in the range of about 0.5% to about 5%.Alternatively, the serum concentration of the second culture medium maybe in the range of about 0.5% to about 2%. Alternatively, the serumconcentration of the second culture medium may be about 2%. Whenculturing pluripotent stem cells with the second culture medium, thetime of culturing may range from about 1 day to about 4 days.

Similar to the first culture medium, Activin A may be used at anyconcentration suitable to cause differentiation of the pluripotent stemcells. The concentration maybe from about 1 pg/ml to about 100 μg/ml. Inan alternate embodiment, the concentration may be about 1 pg/ml to about1 μg/ml. In another alternate embodiment, the concentration may be about1 pg/ml to about 100 ng/ml. In another alternate embodiment, theconcentration may be about 50 ng/ml to about 100 ng/ml. In anotheralternate embodiment, the concentration may be about 100 ng/ml.

The Wnt ligand may be at a concentration of about 1 ng/ml to about 1000ng/ml. In an alternate embodiment, the concentration may be about 10ng/ml to about 100 ng/ml.

The Wnt ligand may be selected from the group consisting of Wnt-1,Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1.In an alternate embodiment, the Wnt ligand is Wnt-3a.

The second culture medium may also contain a GSK-3B inhibitor. TheGSK-3B inhibitor may be added to the first culture medium, to the secondculture medium, or to both the first and second culture media.

The GSK-3B inhibitor may be selected from the group consisting of GSK-3Binhibitor IX and GSK-3B inhibitor XI. In one embodiment, the GSK-3Binhibitor is GSK-3B inhibitor IX.

When culturing pluripotent stem cells with a GSK-3B inhibitor, theconcentration of the GSK-3B inhibitor may be from about 1 nM to about1000 nM. In an alternate embodiment, the pluripotent stem cells arecultured with the GSK-3B inhibitor at a concentration of about 10 nM toabout 100 nM.

Similar to the first culture medium, the second culture medium may alsocontain at least one other additional factor that may enhance theformation of cells expressing markers characteristic of the definitiveendoderm lineage from pluripotent stem cells. Alternatively, the atleast one other additional factor may enhance the proliferation of thecells expressing markers characteristic of the definitive endodermlineage formed by the methods of the present invention. Further, the atleast one other additional factor may enhance the ability of the cellsexpressing markers characteristic of the definitive endoderm lineageformed by the methods of the present invention to form other cell types,or improve the efficiency of any other additional differentiation steps.

The at least one additional factor may be, for example, nicotinamide,members of TGF-β family, including TGF-β1, 2, and 3, serum albumin,members of the fibroblast growth factor family, platelet-derived growthfactor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I,II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagonlike peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody,Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone,aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermalgrowth factor (EGF), gastrin I and II, copper chelators such as, forexample, triethylene pentamine, forskolin, Na-Butyrate, activin,betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid,trichostatin A, sodium butyrate, hepatocyte growth factor (HGF),sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco,CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA),keratinocyte growth factor (KGF), Dickkopf protein family, bovinepituitary extract, islet neogenesis-associated protein (INGAP), Indianhedgehog, sonic hedgehog, proteasome inhibitors, notch pathwayinhibitors, sonic hedgehog inhibitors, or combinations thereof.

The at least one other additional factor may be supplied by conditionedmedia obtained from pancreatic cells lines such as, for example, PANC-1(ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No:CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, forexample, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as,for example, FHs 74 (ATCC No: CCL-241).

Detection of Cells Expressing Markers Characteristic of the DefinitiveEndoderm Linage

Formation of cells expressing markers characteristic of the definitiveendoderm lineage may be determined by testing for the presence of themarkers before and after following a particular protocol. Pluripotentstem cells typically do not express such markers. Thus, differentiationof pluripotent cells is detected when cells begin to express them.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the definitive endoderm lineage.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and ane, UsingAntibodies: A aboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Examples of antibodies useful for detecting certain protein markers arelisted in Table IA. It should be noted that alternate antibodiesdirected to the same markers that are recognized by the antibodieslisted in Table IA are available, or can be readily developed. Suchalternate antibodies can also be employed for assessing expression ofmarkers in the cells isolated in accordance with the present invention.

For example, characteristics of pluripotent stem cells are well known tothose skilled in the art, and additional characteristics of pluripotentstem cells continue to be identified. Pluripotent stem cell markersinclude, for example, the expression of one or more of the following:ABCG2, cripto, FoxD3, Connexin43, Connexin45, Oct4, Sox2, Nanog, hTERT,UTF-1, ZFP42, SSEA-3, SSEA-4, Tra1-60, Tra1-81.

After treating pluripotent stem cells with the methods of the presentinvention, the differentiated cells may be purified by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker, such as CXCR4, expressed bycells expressing markers characteristic of the definitive endodermlineage.

Formation of Cells Expressing Markers Characteristic of the PancreaticEndoderm Lineage

Cells expressing markers characteristic of the definitive endodermlineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endoderm lineage by any method in theart or by any method proposed in this invention.

For example, cells expressing markers characteristic of the definitiveendoderm lineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endoderm lineage according to themethods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401(2006).

For example, cells expressing markers characteristic of the definitiveendoderm lineage are further differentiated into cells expressingmarkers characteristic of the pancreatic endoderm lineage, by treatingthe cells expressing markers characteristic of the definitive endodermlineage with a fibroblast growth factor and the hedgehog signalingpathway inhibitor KAAD-cyclopamine, then removing the medium containingthe fibroblast growth factor and KAAD-cyclopamine and subsequentlyculturing the cells in medium containing retinoic acid, a fibroblastgrowth factor and KAAD-cyclopamine. An example of this method isdisclosed in Nature Biotechnology 24, 1392-1401 (2006).

In one aspect of the present invention, cells expressing markerscharacteristic of the definitive endoderm lineage are furtherdifferentiated into cells expressing markers characteristic of thepancreatic endoderm lineage, by treating the cells expressing markerscharacteristic of the definitive endoderm lineage with retinoic acid andat least one fibroblast growth factor for a period of time. That periodof time may be from about one to about six days.

In an alternate aspect of the present invention, cells expressingmarkers characteristic of the definitive endoderm lineage are furtherdifferentiated into cells expressing markers characteristic of thepancreatic endoderm lineage, by treating the cells with retinoic acidfor a period of time. That period of time maybe from about one to aboutthree days. The retinoic acid is subsequently removed and the cells aretreated with at least one fibroblast growth factor for another period oftime. That period of time may be from about one to about three days.

In one embodiment, the present invention provides a method fordifferentiating cells expressing markers characteristic of thedefinitive endoderm lineage into cells expressing markers characteristicof the pancreatic endoderm lineage, comprising the steps of:

-   -   a. Culturing cells expressing markers characteristic of the        definitive endoderm lineage, and    -   b. Treating the cells expressing markers characteristic of the        definitive endoderm lineage with retinoic acid and at least one        fibroblast growth factor.

Any cell expressing markers characteristic of the definitive endodermlineage is suitable for differentiating into a cell expressing markerscharacteristic of the pancreatic endoderm lineage using this method.

In one embodiment, the cells expressing markers characteristic of thedefinitive endoderm are treated with retinoic acid and at least onefibroblast growth factor for about one to about six days. In oneembodiment, the cells expressing markers characteristic of thedefinitive endoderm are treated with retinoic acid and at least onefibroblast growth factor for about six days.

The at least one fibroblast growth factor is selected from the groupconsisting of FGF-2, FGF-4 and FGF-10.

Any cell expressing markers characteristic of the definitive endodermlineage is suitable for differentiating into a cell expressing markerscharacteristic of the pancreatic endoderm lineage using this method.

In an alternate embodiment, the present invention provides a method fordifferentiating cells expressing markers characteristic of thedefinitive endoderm lineage into cells expressing markers characteristicof the pancreatic endoderm lineage, comprising the steps of:

-   -   a. Culturing cells expressing markers characteristic of the        definitive endoderm lineage,    -   b. Treating the cells expressing markers characteristic of the        definitive endoderm lineage treating the cells with retinoic        acid, and    -   c. Removing the retinoic acid and subsequently treating the        cells with at least one fibroblast growth factor.

Any cell expressing markers characteristic of the definitive endodermlineage is suitable for differentiating into a cell expressing markerscharacteristic of the pancreatic endoderm lineage using this method.

In one embodiment, the cells expressing markers characteristic of thedefinitive endoderm are treated with retinoic acid for about one toabout three days. In one embodiment, the cells expressing markerscharacteristic of the definitive endoderm are treated with retinoic acidfor about three days. In one embodiment, the cells expressing markerscharacteristic of the definitive endoderm are treated with at least onefibroblast growth factor for about one to about three days. In oneembodiment, the cells expressing markers characteristic of thedefinitive endoderm are treated with at least one fibroblast growthfactor for about three days.

The at least one fibroblast growth factor is selected from the groupconsisting of FGF-2, FGF-4 and FGF-10.

Any cell expressing markers characteristic of the definitive endodermlineage is suitable for differentiating into a cell expressing markerscharacteristic of the pancreatic endoderm lineage using this method.

In one embodiment, the cells expressing markers characteristic of thedefinitive endoderm lineage are treated with retinoic acid.Alternatively, the cells expressing markers characteristic of thedefinitive endoderm lineage are treated with FGF-2, or alternativelyFGF-4, or alternatively FGF-10. In an alternate embodiment, the cellsexpressing markers characteristic of the definitive endoderm lineage aretreated with at least one of the following factors: retinoic acid,FGF-2, FGF-4 or FGF-10. In an alternate embodiment, the cells expressingmarkers characteristic of the definitive endoderm lineage are treatedwith retinoic acid and at least one of the following fibroblast growthfactors: FGF-2, FGF-4 or FGF-10. In one embodiment, the cells expressingmarkers characteristic of the definitive endoderm lineage are treatedwith retinoic acid and FGF-2. In another embodiment, the cellsexpressing markers characteristic of the definitive endoderm lineage aretreated with retinoic acid and FGF-4. In a further embodiment, the cellsexpressing markers characteristic of the definitive endoderm lineage aretreated with retinoic acid and FGF-10.

Retinoic acid may be used at a concentration from about 1 nM to about 1mM. In one embodiment, retinoic acid is used at a concentration of 1 μM.

FGF-2 may be used at a concentration from about 50 pg/ml to about 50μg/ml. In one embodiment, FGF-2 is used at a concentration of 50 ng/ml.

FGF-4 may be used at a concentration from about 50 pg/ml to about 50μg/ml. In one embodiment, FGF-4 is used at a concentration of 50 ng/ml.

FGF-10 may be used at a concentration from about 50 pg/ml to about 50μg/ml. In one embodiment, FGF-10 is used at a concentration of 50 ng/ml.

Cells expressing markers characteristic of the definitive endodermlineage may be treated with at least one other additional factor thatmay enhance the formation of cells expressing markers characteristic ofthe pancreatic endoderm lineage. Alternatively, the at least one otheradditional factor may enhance the proliferation of the cells expressingmarkers characteristic of the pancreatic endoderm lineage formed by themethods of the present invention. Further, the at least one otheradditional factor may enhance the ability of the cells expressingmarkers characteristic of the pancreatic endoderm lineage formed by themethods of the present invention to form other cell types, or improvethe efficiency of any other additional differentiation steps.

The at least one additional factor may be, for example, nicotinamide,members of TGF-β family, including TGF-β1, 2, and 3, serum albumin,members of the fibroblast growth factor family, platelet-derived growthfactor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I,II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagonlike peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody,Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone,aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermalgrowth factor (EGF), gastrin I and II, copper chelators such as, forexample, triethylene pentamine, forskolin, Na-Butyrate, activin,betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid,trichostatin A, sodium butyrate, hepatocyte growth factor (HGF),sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco,CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA),keratinocyte growth factor (KGF), Dickkopf protein family, bovinepituitary extract, islet neogenesis-associated protein (INGAP), Indianhedgehog, sonic hedgehog, proteasome inhibitors, notch pathwayinhibitors, sonic hedgehog inhibitors, or combinations thereof.

The at least one other additional factor may be supplied by conditionedmedia obtained from pancreatic cells lines such as, for example, PANC-1(ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No:CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, forexample, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as,for example, FHs 74 (ATCC No: CCL-241).

Detection of Cells Expressing Markers Characteristic of the PancreaticEndoderm Linage

Markers characteristic of the pancreatic endoderm lineage are well knownto those skilled in the art, and additional markers characteristic ofthe pancreatic endoderm lineage continue to be identified. These markerscan be used to confirm that the cells treated in accordance with thepresent invention have differentiated to acquire the propertiescharacteristic of the pancreatic endoderm lineage. Pancreatic endodermlineage specific markers include the expression of one or moretranscription factors such as, for example, Hlxb9, PTF-1a, PDX-1, HNF-6,HNF-1beta.

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the pancreatic endoderm lineage.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Examples of antibodies useful for detecting certain protein markers arelisted in Table IA. It should be noted that alternate antibodiesdirected to the same markers that are recognized by the antibodieslisted in Table IA are available, or can be readily developed. Suchalternate antibodies can also be employed for assessing expression ofmarkers in the cells isolated in accordance with the present invention.

Formation of Cells Expressing Markers of the Pancreatic EndocrineLineage

Cells expressing markers characteristic of the pancreatic endodermlineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage by any method in theart or by any method disclosed in this invention.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage may be differentiated into cells expressing markerscharacteristic of the pancreatic endocrine lineage according to themethods disclosed in D'Amour et al, Nature Biotechnology 24, 1392-1401(2006).

For example, cells expressing markers characteristic of the pancreaticendoderm lineage are further differentiated into cells expressingmarkers characteristic of the pancreatic endocrine lineage, by culturingthe cells expressing markers characteristic of the pancreatic endodermlineage in medium containing DAPT and exendin 4, then removing themedium containing DAPT and exendin 4 and subsequently culturing thecells in medium containing exendin 1, IGF-1 and HGF. An example of thismethod is disclosed in Nature Biotechnology 24, 1392-1401 (2006).

For example, cells expressing markers characteristic of the pancreaticendoderm lineage are further differentiated into cells expressingmarkers characteristic of the pancreatic endocrine lineage, by culturingthe cells expressing markers characteristic of the pancreatic endodermlineage in medium containing exendin 4, then removing the mediumcontaining exendin 4 and subsequently culturing the cells in mediumcontaining exendin 1, IGF-1 and HGF. An example of this method isdisclosed in D'Amour et al, Nature Biotechnology, 2006.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage are further differentiated into cells expressingmarkers characteristic of the pancreatic endocrine lineage, by culturingthe cells expressing markers characteristic of the pancreatic endodermlineage in medium containing DAPT and exendin 4. An example of thismethod is disclosed in D'Amour et al, Nature Biotechnology, 2006.

For example, cells expressing markers characteristic of the pancreaticendoderm lineage are further differentiated into cells expressingmarkers characteristic of the pancreatic endocrine lineage, by culturingthe cells expressing markers characteristic of the pancreatic endodermlineage in medium containing exendin 4. An example of this method isdisclosed in D'Amour et al, Nature Biotechnology, 2006.

In one aspect of the present invention, cells expressing markerscharacteristic of the pancreatic endoderm lineage are furtherdifferentiated into cells expressing markers characteristic of thepancreatic endocrine lineage, by treating the cells expressing markerscharacteristic of the pancreatic endoderm lineage with a factor thatinhibits the Notch signaling pathway. The factor that inhibits the Notchsignaling pathway may be an antagonist for the Notch extracellularreceptor. Alternatively, the factor may inhibit the biological activityof the Notch receptor. Alternatively, the factor may inhibit or be anantagonist of an element in the Notch signal transduction pathway withina cell.

In one embodiment the factor that inhibits the Notch signaling pathwayis a γ-secretase inhibitor. In one embodiment, the γ-secretase inhibitoris1SBenzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-52Rhydrozy-5 phenylpentyl] carbamic Acid tert-butyl Ester, also known asL-685,458.

L-685,458 may be used at a concentration from about 0.1 μM to about 100μM. In one embodiment, L-685,458 is used at a concentration of about 90μM. In one embodiment, L-685,458 is used at a concentration of about 80μM. In one embodiment, L-685,458 is used at a concentration of about 70μM. In one embodiment, L-685,458 is used at a concentration of about 60μM. In one embodiment, L-685,458 is used at a concentration of about 50μM. In one embodiment, L-685,458 is used at a concentration of about 40μM. In one embodiment, L-685,458 is used at a concentration of about 30μM. In one embodiment, L-685,458 is used at a concentration of about 20μM. In one embodiment, L-685,458 is used at a concentration of about 10μM.

In one embodiment, the present invention provides a method fordifferentiating cells expressing markers characteristic of thepancreatic endoderm lineage into cells expressing markers characteristicof the pancreatic endocrine lineage, comprising the steps of:

-   -   a. Culturing cells expressing markers characteristic of the        pancreatic endoderm lineage, and    -   b. Treating the cells with a factor that inhibits the Notch        signaling pathway.

Any cell expressing markers characteristic of the pancreatic endodermlineage is suitable for differentiating into a cell expressing markerscharacteristic of the pancreatic endocrine lineage using this method.

In one embodiment, factor that inhibits the Notch signaling pathway is aγ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl]carbamic Acid tert-butyl Ester, also known as L-685,458.

The cells expressing markers characteristic of the pancreatic endodermlineage are treated with the factor that inhibits the Notch signalingpathway for about one to about five days. Alternatively, the cellsexpressing markers characteristic of the pancreatic endoderm lineage aretreated with the factor that inhibits the Notch signaling pathway forabout three to about five days. Alternatively, the cells expressingmarkers characteristic of the pancreatic endoderm lineage are treatedwith the factor that inhibits the Notch signaling pathway for about fivedays.

In one embodiment, factor that inhibits the Notch signaling pathway is aγ-secretase inhibitor. In one embodiment, the γ-secretase inhibitor is1S-Benzyl-4R-[1-(1S-carbamoyl-2-phenethylcarbamoyl)-1S-3-methylbutylcarbamoyl]-2R-hydrozy-5-phenylpentyl]carbamic Acid tert-butyl Ester, also known as L-685,458.

L-685,458 may be used at a concentration from about 0.1 μM to about 100μM. In one embodiment, L-685,458 is used at a concentration of about 90μM. In one embodiment, L-685,458 is used at a concentration of about 80μM. In one embodiment, L-685,458 is used at a concentration of about 70μM. In one embodiment, L-685,458 is used at a concentration of about 60μM. In one embodiment, L-685,458 is used at a concentration of about 50μM. In one embodiment, L-685,458 is used at a concentration of about 40μM. In one embodiment, L-685,458 is used at a concentration of about 30μM. In one embodiment, L-685,458 is used at a concentration of about 20μM. In one embodiment, L-685,458 is used at a concentration of about 10μM.

Cells expressing markers characteristic of the pancreatic endodermlineage may be treated with at least one other additional factor thatmay enhance the formation of cells expressing markers characteristic ofthe pancreatic endocrine lineage. Alternatively, the at least one otheradditional factor may enhance the proliferation of the cells expressingmarkers characteristic of the pancreatic endocrine lineage formed by themethods of the present invention. Further, the at least one otheradditional factor may enhance the ability of the cells expressingmarkers characteristic of the pancreatic endocrine lineage formed by themethods of the present invention to form other cell types, or improvethe efficiency of any other additional differentiation steps.

The at least one additional factor may be, for example, nicotinamide,members of TGF-β family, including TGF-β1, 2, and 3, serum albumin,members of the fibroblast growth factor family, platelet-derived growthfactor-AA, and -BB, platelet rich plasma, insulin growth factor (IGF-I,II), growth differentiation factor (GDF-5, -6, -8, -10, 11), glucagonlike peptide-I and II (GLP-I and II), GLP-1 and GLP-2 mimetobody,Exendin-4, retinoic acid, parathyroid hormone, insulin, progesterone,aprotinin, hydrocortisone, ethanolamine, beta mercaptoethanol, epidermalgrowth factor (EGF), gastrin I and II, copper chelators such as, forexample, triethylene pentamine, forskolin, Na-Butyrate, activin,betacellulin, ITS, noggin, neurite growth factor, nodal, valporic acid,trichostatin A, sodium butyrate, hepatocyte growth factor (HGF),sphingosine-1, VEGF, MG132 (EMD, CA), N2 and B27 supplements (Gibco,CA), steroid alkaloid such as, for example, cyclopamine (EMD, CA),keratinocyte growth factor (KGF), Dickkopf protein family, bovinepituitary extract, islet neogenesis-associated protein (INGAP), Indianhedgehog, sonic hedgehog, proteasome inhibitors, notch pathwayinhibitors, sonic hedgehog inhibitors, or combinations thereof.

The at least one other additional factor may be supplied by conditionedmedia obtained from pancreatic cells lines such as, for example, PANC-1(ATCC No: CRL-1469), CAPAN-1 (ATCC No: HTB-79), BxPC-3 (ATCC No:CRL-1687), HPAF-II (ATCC No: CRL-1997), hepatic cell lines such as, forexample, HepG2 (ATCC No: HTB-8065), and intestinal cell lines such as,for example, FHs 74 (ATCC No: CCL-241).

Detection of Cells Expressing Markers Characteristic of the PancreaticEndocrine Linage

Markers characteristic of cells of the pancreatic endocrine lineage arewell known to those skilled in the art, and additional markerscharacteristic of the pancreatic endocrine lineage continue to beidentified. These markers can be used to confirm that the cells treatedin accordance with the present invention have differentiated to acquirethe properties characteristic of the pancreatic endocrine lineage.Pancreatic endocrine lineage specific markers include the expression ofone or more transcription factors such as, for example, NGN-3, NeuroD,Islet-1.

Markers characteristic of cells of the β cell lineage are well known tothose skilled in the art, and additional markers characteristic of the βcell lineage continue to be identified. These markers can be used toconfirm that the cells treated in accordance with the present inventionhave differentiated to acquire the properties characteristic of theβ-cell lineage. β cell lineage specific characteristics include theexpression of one or more transcription factors such as, for example,Pdx1 (pancreatic and duodenal homeobox gene-1), Nkx2.2, Nkx6.1, Isl1,Pax6, Pax4, NeuroD, Hnf1b, Hnf-6, Hnf-3beta, and MafA, among others.These transcription factors are well established in the art foridentification of endocrine cells. See, e.g., Edlund (Nature ReviewsGenetics 3: 524-632 (2002)).

The efficiency of differentiation may be determined by exposing atreated cell population to an agent (such as an antibody) thatspecifically recognizes a protein marker expressed by cells expressingmarkers characteristic of the pancreatic endocrine lineage.Alternatively, the efficiency of differentiation may be determined byexposing a treated cell population to an agent (such as an antibody)that specifically recognizes a protein marker expressed by cellsexpressing markers characteristic of the β cell lineage.

Methods for assessing expression of protein and nucleic acid markers incultured or isolated cells are standard in the art. These includequantitative reverse transcriptase polymerase chain reaction (RT-PCR),Northern blots, in situ hybridization (see, e.g., Current Protocols inMolecular Biology (Ausubel et al., eds. 2001 supplement)), andimmunoassays such as immunohistochemical analysis of sectioned material,Western blotting, and for markers that are accessible in intact cells,flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, UsingAntibodies: A Laboratory Manual, New York: Cold Spring Harbor LaboratoryPress (1998)).

Examples of antibodies useful for detecting certain protein markers arelisted in Table IA. It should be noted that alternate antibodiesdirected to the same markers that are recognized by the antibodieslisted in Table IA are available, or can be readily developed. Suchalternate antibodies can also be employed for assessing expression ofmarkers in the cells isolated in accordance with the present invention.

Therapies

In one aspect, the present invention provides a method for treating apatient suffering from, or at risk of developing, Type1 diabetes. Thismethod involves culturing pluripotent stem cells, differentiating thepluripotent stem cells in vitro into a n-cell lineage, and implantingthe cells of a β-cell lineage into a patient.

In yet another aspect, this invention provides a method for treating apatient suffering from, or at risk of developing, Type 2 diabetes. Thismethod involves culturing pluripotent stem cells, differentiating thecultured cells in vitro into a β-cell lineage, and implanting the cellsof a β-cell lineage into the patient.

If appropriate, the patient can be further treated with pharmaceuticalagents or bioactives that facilitate the survival and function of thetransplanted cells. These agents may include, for example, insulin,members of the TGF-β family, including TGF-β1, 2, and 3, bonemorphogenic proteins (BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13),fibroblast growth factors-1 and -2, platelet-derived growth factor-AA,and -BB, platelet rich plasma, insulin growth factor (IGF-I, II) growthdifferentiation factor (GDF-5, -6, -7, -8, -10, -15), vascularendothelial cell-derived growth factor (VEGF), pleiotrophin, endothelin,among others. Other pharmaceutical compounds can include, for example,nicotinamide, glucagon like peptide-I (GLP-1) and II, GLP-1 and 2mimetibody, Exendin-4, retinoic acid, parathyroid hormone, MAPKinhibitors, such as, for example, compounds disclosed in U.S. PublishedApplication 2004/0209901 and U.S. Published Application 2004/0132729.

The pluripotent stem cells may be differentiated into aninsulin-producing cell prior to transplantation into a recipient. In aspecific embodiment, the pluripotent stem cells are fully differentiatedinto β-cells, prior to transplantation into a recipient. Alternatively,the pluripotent stem cells may be transplanted into a recipient in anundifferentiated or partially differentiated state. Furtherdifferentiation may take place in the recipient.

Definitive endoderm cells or, alternatively, pancreatic endoderm cells,or, alternatively, β cells, may be implanted as dispersed cells orformed into clusters that may be infused into the hepatic portal vein.Alternatively, cells may be provided in biocompatible degradablepolymeric supports, porous non-degradable devices or encapsulated toprotect from host immune response. Cells may be implanted into anappropriate site in a recipient. The implantation sites include, forexample, the liver, natural pancreas, renal subcapsular space, omentum,peritoneum, subserosal space, intestine, stomach, or a subcutaneouspocket.

To enhance further differentiation, survival or activity of theimplanted cells, additional factors, such as growth factors,antioxidants or anti-inflammatory agents, can be administered before,simultaneously with, or after the administration of the cells. Incertain embodiments, growth factors are utilized to differentiate theadministered cells in vivo. These factors can be secreted by endogenouscells and exposed to the administered cells in situ. Implanted cells canbe induced to differentiate by any combination of endogenous andexogenously administered growth factors known in the art.

The amount of cells used in implantation depends on a number of variousfactors including the patient's condition and response to the therapy,and can be determined by one skilled in the art.

In one aspect, this invention provides a method for treating a patientsuffering from, or at risk of developing diabetes. This method involvesculturing pluripotent stem cells, differentiating the cultured cells invitro into a β-cell lineage, and incorporating the cells into athree-dimensional support. The cells can be maintained in vitro on thissupport prior to implantation into the patient. Alternatively, thesupport containing the cells can be directly implanted in the patientwithout additional in vitro culturing. The support can optionally beincorporated with at least one pharmaceutical agent that facilitates thesurvival and function of the transplanted cells.

Support materials suitable for use for purposes of the present inventioninclude tissue templates, conduits, barriers, and reservoirs useful fortissue repair. In particular, synthetic and natural materials in theform of foams, sponges, gels, hydrogels, textiles, and nonwovenstructures, which have been used in vitro and in vivo to reconstruct orregenerate biological tissue, as well as to deliver chemotactic agentsfor inducing tissue growth, are suitable for use in practicing themethods of the present invention. See, for example, the materialsdisclosed in U.S. Pat. Nos. 5,770,417, 6,022,743, 5,567,612, 5,759,830,6,626,950, 6,534,084, 6,306,424, 6,365,149, 6,599,323, 6,656,488, U.S.Published Application 2004/0062753 A1, U.S. Pat. Nos. 4,557,264 and6,333,029.

To form a support incorporated with a pharmaceutical agent, thepharmaceutical agent can be mixed with the polymer solution prior toforming the support. Alternatively, a pharmaceutical agent could becoated onto a fabricated support, preferably in the presence of apharmaceutical carrier. The pharmaceutical agent may be present as aliquid, a finely divided solid, or any other appropriate physical form.Alternatively, excipients may be added to the support to alter therelease rate of the pharmaceutical agent. In an alternate embodiment,the support is incorporated with at least one pharmaceutical compoundthat is an anti-inflammatory compound, such as, for example compoundsdisclosed in U.S. Pat. No. 6,509,369.

The support may be incorporated with at least one pharmaceuticalcompound that is an anti-apoptotic compound, such as, for example,compounds disclosed in U.S. Pat. No. 6,793,945.

The support may also be incorporated with at least one pharmaceuticalcompound that is an inhibitor of fibrosis, such as, for example,compounds disclosed in U.S. Pat. No. 6,331,298.

The support may also be incorporated with at least one pharmaceuticalcompound that is capable of enhancing angiogenesis, such as, forexample, compounds disclosed in U.S. Published Application 2004/0220393and U.S. Published Application 2004/0209901.

The support may also be incorporated with at least one pharmaceuticalcompound that is an immunosuppressive compound, such as, for example,compounds disclosed in U.S. Published Application 2004/0171623.

The support may also be incorporated with at least one pharmaceuticalcompound that is a growth factor, such as, for example, members of theTGF-β family, including TGF-β1, 2, and 3, bone morphogenic proteins(BMP-2, -3, -4, -5, -6, -7, -11, -12, and -13), fibroblast growthfactors-1 and -2, platelet-derived growth factor-AA, and -BB, plateletrich plasma, insulin growth factor (IGF-I, II) growth differentiationfactor (GDF-5, -6, -8, -10, -15), vascular endothelial cell-derivedgrowth factor (VEGF), pleiotrophin, endothelin, among others. Otherpharmaceutical compounds can include, for example, nicotinamide, hypoxiainducible factor 1-alpha, glucagon like peptide-I (GLP-1), GLP-1 andGLP-2 mimetibody, and II, Exendin-4, nodal, noggin, NGF, retinoic acid,parathyroid hormone, tenascin-C, tropoelastin, thrombin-derivedpeptides, cathelicidins, defensins, laminin, biological peptidescontaining cell- and heparin-binding domains of adhesive extracellularmatrix proteins such as fibronectin and vitronectin, MAPK inhibitors,such as, for example, compounds disclosed in U.S. Published Application2004/0209901 and U.S. Published Application 2004/0132729.

The incorporation of the cells of the present invention into a scaffoldcan be achieved by the simple depositing of cells onto the scaffold.Cells can enter into the scaffold by simple diffusion (J. Pediatr. Surg.23 (1 Pt 2): 3-9 (1988)). Several other approaches have been developedto enhance the efficiency of cell seeding. For example, spinner flaskshave been used in seeding of chondrocytes onto polyglycolic acidscaffolds (Biotechnol. Prog. 14(2): 193-202 (1998)). Another approachfor seeding cells is the use of centrifugation, which yields minimumstress to the seeded cells and enhances seeding efficiency. For example,Yang et al. developed a cell seeding method (J. Biomed. Mater. Res.55(3): 379-86 (2001)), referred to as Centrifugational CellImmobilization (CCI).

The present invention is further illustrated, but not limited by, thefollowing examples.

EXAMPLES Example 1 Human Embryonic Stem Cell Culture

The human embryonic stem cell lines H1, H7 and H9 were obtained fromWiCell Research Institute, Inc., (Madison, WI) and cultured according toinstructions provided by the source institute. Briefly, cells werecultured on mouse embryonic fibroblast (MEF) feeder cells in ES cellmedium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20%knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mMbeta-mercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basicfibroblast growth factor (bFGF) (all from Invitrogen/GIBCO). MEF cells,derived from E13 to 13.5 mouse embryos, were purchased from CharlesRiver. MEF cells were expanded in DMEM medium supplemented with 10% FBS(Hyclone), 2 mM glutamine, and 100 mM MEM nonessential amino acids.Sub-confluent MEF cell cultures were treated with 10 μg/ml mitomycin C(Sigma, St. Louis, MO) for 3 h to arrest cell division, then trypsinizedand plated at 2×10⁴/cm² on 0.1% bovine gelatin-coated dishes. MEF cellsfrom passage two through four were used as feeder layers. Humanembryonic stem cells plated on MEF cell feeder layers were cultured at37° C. in an atmosphere of 5% CO₂/within a humidified tissue cultureincubator. When confluent (approximately 5-7 days after plating), humanembryonic stem cells were treated with 1 mg/ml collagenase type IV(Invitrogen/GIBCO) for 5-10 min and then gently scraped off the surfaceusing a 5-ml pipette. Cells were spun at 900 rpm for 5 min, and thepellet was resuspended and re-plated at a 1:3 to 1:4 ratio of cells infresh culture medium.

Example 2 Formation of Definitive Endoderm

The effects of activin A on the expression of markers of definitiveendoderm were examined. Activin A (100 ng/ml) was added to populationsof human embryonic stem cells cultured on mouse embryonic fibroblasts.Cells were cultured continuously in the presence of activin A andharvested at the times indicated. The level of expression of definitiveendoderm markers was examined by PCR (FIGS. 1A and 1B), FACS (resultssummarized in Table II), and immunohistochemistry (FIGS. 2A, 2B and 2C).

Activin A evoked a time-dependent increase in the expression of CXCR4,GATA4, HNF-3beta, Mixl1 and Sox-17 mRNA in the H9 line (FIG. 1A). Asignificant up regulation of anterior endoderm markers, Cerberus, Otx-1and Hex genes was also observed (FIG. 1B). An increase in CXCR4 proteinwas observed by FACS analysis following activin A treatment. Theexpression of E-cadherin and N-cadherin did not change following activinA treatment (Table IIA). CXCR4 positive cells were also highly positivefor C-kit, EPCAM, CD99, and negative for CD9. The expression pattern forthese markers was consistent among all three hES cell lines examined(Table IIB for H7 and Table IIC for H1). Immunocytochemistry conductedon cells treated with activin A for five days revealed that 30-40% cellsin the treated culture were positive for Sox17 and HNF-3beta. Inparallel, almost 100% of the differentiated cells were still Oct4positive (FIGS. 2A, 2B and 2C). With the decrease in expression ofsurface markers of pluripotency, combined with an increase in theexpression of definitive endoderm markers, these data suggest thatactivin A promotes the differentiation of human embryonic stem cells todefinitive endoderm.

Example 3 Formation of Pancreatic Endoderm

Growth factors known to induce the differentiation of human embryonicstem cells to pancreatic endoderm were added to cell cultures. Inparticular, activin A, bFGF, and retinoic acid, known to induce theformation of pancreatic endoderm, were added to cell cultures.

In a first series of experiments, activin A, was added to populations ofhuman embryonic stem cells cultured on mouse embryonic fibroblasts forup to seven days in DMEM/F12 supplemented with 0% to 2% serum andActivin A (100 ng/ml). Cells were harvested at the time points indicatedin FIGS. 3A, 3B, 3C and 3D and assayed by PCR for the expression ofgenes shown (FIGS. 3A, 3B, 3C, 3D, 4A, 4B, 5A and 5B). In FIGS. 3A, 3B,3C and 3D, PCR analysis indicated that activin treated cells expressed abroad spectrum of genes associated with endoderm development, includingGATA4 (FIG. 3A), Sox-17 (FIG. 3B), HNF-3beta (FIG. 3C), and Mixl-1 (FIG.3D). However, no Pdx1 gene expression was observed. The same expressionpattern of endoderm lineage markers was observed in Activin A treated H7cells (FIGS. 6A, 6B, 6C, 6D, 6E and 6F). At this stage, there was nosignificant decrease of Oct4 expression.

Activin A evoked a time-dependent decrease in the expression of theextraembryonic endoderm markers Sox7 (FIG. 4A) and AFP (FIG. 4B).Activin A decreased the expression of Brachyury (FIG. 5A) but had noeffect on expression of the neuronal marker Zic1 (FIG. 5B).

Taken together, these data suggest that the increased expression ofSox-17, Mixl1, Gata4, and HNF-3beta together with the upregulation ofanterior endoderm markers Otx1, Cer1 and Hex genes, corresponds to theformation of definitive endoderm in response to activin A treatment.Analysis of definitive endoderm markers by immunocytochemistry revealedthat protein expression for these genes also reflected the trendsobserved in mRNA expression. Levels of expression for HNF-3beta, Sox-17,and GATA4 were low in untreated cells, approximately 10 to 20% of allcells. Activin A (100 ng/ml) treatment for five days increased theexpression of HNF-3beta, Sox-17, and GATA4 to approximately 50% to 90%of all cells (FIGS. 7A, 7B, 7C, 7D, 7E and 7F).

In a second series of experiments, cultures of human embryonic stemcells were maintained in undifferentiated culture conditions for 2-3days according to the methods described in Example 1. After the cellswere 70-80% confluent, the medium was changed to DMEM/F12 with 0 to 2%FBS with addition of activin A at 100 ng/ml and cultured in the presenceof activin A for either three, five, or seven days. After this timeinterval, the cells were then further treated for five to six days withcombinations of retinoic acid and bFGF as shown in FIGS. 8A, 8B and 8C.Cultures were harvested and samples of mRNA were collected for analysis.Control cultures consisting of cells treated with activin A alone forfive days were also included.

Gene expression analysis revealed that activin A or retinoic acid alonedid not induce the expression of Pdx1. Similar results were observed incultures of cells treated with retinoic acid in combination with FGF andin the presence of activin A (FIG. 8A). However, treatment of cells withretinoic acid and FGF in the absence of activin A increased theexpression of Pdx1 still further (FIG. 8A). Cells treated for three dayswith activin A, then treated for 5 days with 1 μM retinoic acid and 50ng/ml bFGF (also known as FGF-2) in the absence of activin A showed alevel of Pdx1 expression that was approximately 3500-fold higher thanthat observed in samples with activin A treatment alone for 5 days (FIG.8A). Immunocytochemistry showed that 5 to 20% of all cells expressedPdx1 (FIGS. 9A and 9B).

Treatment with 1 μM retinoic acid and bFGF in the absence of activin Aalso caused an increase in the expression of GLUT-2 and PTF1a (FIG. 8C)that was not observed in cells treated in the presence of activin Aalone. The largest increase in expression of GLUT-2 and PTF1a wasobserved in cells treated with 1 μM retinoic acid and 50 ng/ml bFGF.Taken together, these data suggest that the formation of pancreaticendoderm is further enhanced by removal of activin A from cell culturesafter definitive endoderm has been formed.

Example 4 Formation of Pancreatic Endocrine Cells

Cultures of human embryonic stem cells were maintained inundifferentiated culture conditions for 3-4 days according to themethods described in Example 1. After the cells were 50-60% confluent,the medium was changed to DMEM/F12 without FBS, containing activin A at100 ng/ml, and the cells were cultured in this medium for one day.Following the one day culture, the medium was removed and replaced withmedium containing 0.5% FBS with 100 ng/ml activin A, and the cells werecultured for one day. Following the second one-day culture, the mediumwas removed and replaced with medium containing 2% FBS with 100 ng/mlactivin A, and the cells were cultured for one day. After this timeinterval, the cells were then treated for six days with combinations ofretinoic acid and FGF as outlined in Example 2, then the culture mediumwas removed and replaced with medium comprising DMEM/F12 with 2% FBS,containing the γ-secretase inhibitors L-685,458 at 10 μM for three days.Cultures were harvested and samples of mRNA were collected for analysis.Control cultures consisting of cells treated with activin A alone forfive days were also included.

Gene expression analysis revealed that activin A alone or in combinationwith retinoic acid and FGFs did not induce the expression of Ngn3 orInsulin (FIGS. 10A and 10C). A decrease in the expression of Hes-1 wasalso observed following treatment with L-685,458. The maximal inhibitionwas observed on day three post treatment (FIG. 10D). However, treatmentof cells with L-685,458 induced the expression of Ngn3 to a levelapproximately higher than that observed in samples treated with activinA alone or retinoic acid with FGFs in combination. A 70-fold increase ofinsulin expression was observed in samples treated with the γ-secretaseinhibitor. NeuroD1 expression was also increased further by theL-685,458 treatment (FIG. 10A). Taken together, these data suggest thatthe formation of endocrine cells is further enhanced by removal ofretinoic acid and FGFs from cell culture and the addition of γ-secretaseinhibitors after pancreatic endoderm has been formed.

Example 5 Formation of Pancreatic Endocrine Cells Expressing Nkx2.2

Definitive endoderm cells obtained according to the methods outlined inExample 2 were treated as follows: Cells were cultured in basal medium,comprising DMEM/F12 with 2% FBS plus 50 ng/ml activin A, 50 ng/ml basicFGF and 1 μM of Retinoic Acid for 3 to 5 days. Cells were continuouslycultured for another 3 to 5 days in basal medium with retinoic acid at 1μM, alone or with bFGF. RNA samples were harvested from cells at varioustime points along this process to help evaluate the directeddifferentiation of the cells. Furthermore, culture medium and factorswere regularly removed and replenished throughout the differentiationprotocol. Addition of activin A showed an increase of Nkx2.2 expressionabout 35-fold compared to samples without activin A. Samples treatedwith activin A for the first three days of culture maintained Pdx1expression at a level similar to samples containing no activin A (FIGS.11A and 11B). Taken together, these data suggest that the expression ofthe pancreatic endocrine marker Nkx2.2 is further enhanced by addingActivin A to the first three days of retinoic acid and bFGF treatment.

Example 6 Passage and Expansion of Pancreatic Endoderm Cells in Culture

This example demonstrates that pancreatic endoderm cells derived fromhuman embryonic stem cells herein can be maintained in cell culture andpassaged without further differentiation. Pancreatic endoderm cells weredifferentiated in the presence of 100 ng/ml activin A in low serumDMEM/F12. The low serum DMEM/F12 contained 0% (v/v) fetal bovine serum(FBS) on day 1, 0.5% (v/v) FBS on day two and 2% (v/v) FBS on each daythereafter. After four days of differentiation, the cells were culturedin low serum DMEM/F12 contained 2% (v/v) FBS, 1 μM retinoic acid and 50ng/ml bFGF for a total of six more days. After the six days ofdifferentiation, the cells were maintained in culture in low serumDMEM/F12 contained 2% (v/v) FBS in the presence of 50 ng/ml FGF10 for atotal of 6 days. During the six-day culture period, the pancreaticendoderm cells were passaged twice and cell population-doubling time isabout 36 to 48 hours during this 6-day culture. On days 0, 3, and 6 ofculture, Q-PCR was used to measure the expression of marker genesindicative of pancreatic endoderm. FIG. 12 shows that cells grown in thepresence of 50 ng/ml FGF10 maintained expression of the pancreaticendoderm marker Pdx1 during the 6 day culture period subsequent to theirderivation.

Example 7 Derivation of Hepatocytes from Human Embryonic Stem Cells

Cultures of human embryonic stem cells were maintained inundifferentiated culture conditions for 2-3 days according to themethods described in Example 1. After cells were 70-80% confluent, themedium was changed to DMEM/F12 with 2% FBS containing activin A at 100ng/ml, and cells were cultured in the presence of activin A for sevendays. After 7 days treatment with activin A, the cells were then treatedfor five days with the conditions shown in FIGS. 13A and 13B. After thistime, the cells were harvested, and samples of mRNA were collected foranalysis.

An increase in the expression of α-fetoprotein (AFP) and albumin wasobserved (FIG. 13A) for cells cultured in the absence of activin A. Thiswas further increased by retinoic acid and FGF-4 (FIG. 13B). Takentogether, these data suggest that cultures of human embryonic stem cellsare capable of expressing hepatocyte markers following the treatmentdescribed above. Furthermore, human embryonic stem cells are capable ofbeing differentiated into cells expressing markers that arecharacteristic of hepatocytes.

Example 8 Characterization of the H9 Human Embryonic Stem Cell Line

The quality of H9 cells was monitored over time by evaluating expressionof several markers expressed by undifferentiated ES cells (Carpenter etal., 2001; Reubinoff et al., 2000; Thomson et al., 1998a). H9 cellsexhibited reciprocal expression of stage-specific embryonic antigens(Table III). H9 cells play strong immunoreactivity for SSEA-3, SSEA-4,Tra-1-60, Tra-1-81, AP and CD9 antigens, all of which are characteristicof undifferentiated human embryonic stem cells.

RT-PCR was performed to assess the expression of genes characteristic ofembryonic stem cells, such as, for example, OCT3/4, SOX-2, UTF-1, REX-1,Cx43, Cx45, ABCG-2 and TERT, confirming that the cells grown in thisexample appeared similar to previously described undifferentiatedembryonic stem cells (Table III). OCT3/4 protein expression and AlkalinePhosphatase activity (Chemicon) were confirmed by immunostaining. Amajority of H9 cells were positive for OCT3/4 and AP (FIGS. 14A and14B). Overall, these results demonstrate that the H9 cells used in thisexample were not significantly different in morphology, antigenimmunostaining, or pluripotency marker expression when compared toreports from other laboratories.

Example 9 Fluorescence-Activated Cell Sorting (FACS) Analysis

Adhered cells were removed from culture plates by five-minute incubationwith TrypLE™ Express solution (Invitrogen, CA). Released cells wereresuspended in human embryonic stem cell culture medium and recovered bycentrifugation, followed by washing and resuspending the cells in astaining buffer consisting of 2% BSA, 0.05% sodium azide in PBS (Sigma,MO). As appropriate, the cells were Fc-receptor blocked for 15 minutesusing a 0.1% γ-globulin (Sigma) solution. Aliquots (approximately 10⁵cells) were incubated with either phycoerythirin (PE) or allophycocyanin(APC) conjugated monoclonal antibodies (5 μl antibody per 10⁶ cells), asindicated in Table I, or with an unconjugated primary antibody. Controlsincluded appropriate isotype matched antibodies, unstained cells, andcells stained only with secondary conjugated antibody. All incubationswith antibodies were performed for 30 mins at 4° C. after which thecells were washed with the staining buffer. Samples that were stainedwith unconjugated primary antibodies were incubated for an additional 30mins at 4° C. with secondary conjugated PE or -APC labeled antibodies.See Table I for a list of secondary antibodies used. Washed cells werepelleted and resuspended in the staining buffer, and the cell surfacemolecules were identified using a FACS Array (BD Biosciences)instrument, collecting at least 10,000 events.

Example 10 Immunocytochemistry

Cells seeded on 0.1% Matrigel (BD) coated dishes were fixed with 4%paraformaldheyde for 20 min at room temperature. Fixed cells wereblocked for 1 h at room temperature with PBS/0.1% BSA/10% normal chickserum/0.5% Triton X-100 and then incubated overnight with primaryantibodies in PBS/0.1% BSA/10% normal chick serum at 4° C. The list ofprimary antibodies and their working dilutions are shown in Table 1B.After three washes in PBS/0.1% BSA, fluorescent secondary antibodies ata 1:100 dilution in PBS were incubated with cells for 1 h at roomtemperature to allow binding. Control samples included reactions wherethe primary antibody was omitted or where the primary antibody wasreplaced with corresponding matched negative control immunoglobulins atthe same concentration as the primary antibodies. Stained samples wererinsed; a drop of PROLONG® (Invitrogen, CA) containingdiamidino-2-phenylindole, dihydrochloride (DAPI) was added to eachsample to counter-stain the nucleus and to function as an anti-fadereagent. Images were acquired using a Nikon Confocal Eclipse C-1inverted microscope (Nikon, Japan) and a 10-60× objective.

Example 11 PCR Analysis of Undifferentiated Cells

RNA extraction, purification, and cDNA synthesis. RNA samples werepurified by binding to a silica-gel membrane (Rneasy Mini Kit, Qiagen,CA) in the presence of an ethanol-containing, high-salt buffer followedby washing to remove contaminants. The RNA was further purified using aTURBO DNA-free kit (Ambion, INC), and high-quality RNA was then elutedin water. Yield and purity were assessed by A260 and A280 readings on aspectrophotometer. cDNA copies were made from purified RNA using an ABI(ABI, CA) high capacity cDNA archive kit.

Real-time PCR amplification and quantitative analysis. Unless otherwisestated, all reagents were purchased from Applied Biosystems. Real-timePCR reactions were performed using the ABI PRISM® 7900 SequenceDetection System. TAQMAN® UNIVERSAL PCR MASTER MIX® (ABI, CA) was usedwith 20 ng of reverse transcribed RNA in a total reaction volume of 20pd. Each cDNA sample was run in duplicate to correct for pipettingerrors. Primers and FAM-labeled TAQMAN® probes were used atconcentrations of 200 nM. The level of expression for each target genewas normalized using a human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) endogenous control previously developed by Applied Biosystem.Primer and probe sets are listed as follows: Oct3/4 (Hs00742896), SOX-2(Hs00602736), UTF-1 (Hs00747497), Rex-1 (Hs00399279), Connexin 43(Hs00748445), Connexin (Hs00271416), ABCG2 (Hs00184979), Otx-1(Hs00222238), Cerberus (Hs00193796)m, Albumin (Hs00609411).

After an initial incubation at 50° C. for 2 min followed by 95° C. for10 min, samples were cycled 40 times in two stages—a denaturation stepat 95° C. for 15 sec followed by an annealing/extension step at 60° C.for 1 min. Data analysis was carried out using GENEAMP®7000 SequenceDetection System software. For each primer/probe set, a C_(t) value wasdetermined as the cycle number at which the fluorescence intensityreached a specific value in the middle of the exponential region ofamplification. Relative gene expression levels were calculated using thecomparative C_(t) method. Briefly, for each cDNA sample, the endogenouscontrol C_(t) value was subtracted from the gene of interest C_(t) togive the delta C_(t) value (ΔC_(t)). The normalized amount of target wascalculated as 2^(−ΔCt), assuming amplification to be 100% efficiency.Final data were expressed relative to a calibrator sample.

Example 12 Karyotype Analysis

The karyotype of H9 cells was determined by standard G-banding karyotypeanalysis. A total of 100 metaphase spreads were evaluated (AppliedGenetics Laboratories, Inc.). No chromosome aberrations were found in100 cells analyzed. Cytogenetic analysis showed that the cells had anormal number of autosomes and a modal chromosome number of 46. FIG. 15depicts a typical karyotype obtained from the human embryonic stem cellline H9.

Example 13 Human Embryonic Stem Cell Culture on Tissue Culture SubstrateCoated with Extracellular Matrix

The human embryonic stem cell lines H1, H7, and H9 were obtained fromWiCell Research Institute, Inc., (Madison, WI) and cultured according toinstructions provided by the source institute. Briefly, cells werecultured on mouse embryonic fibroblast (MEF) feeder cells in ES cellmedium consisting of DMEM/F12 (Invitrogen/GIBCO) supplemented with 20%knockout serum replacement, 100 nM MEM nonessential amino acids, 0.5 mMbeta-mercaptoethanol, 2 mM L-glutamine with 4 ng/ml human basicfibroblast growth factor (bFGF). MEF cells, derived from E13 to 13.5mouse embryos, were purchased from Charles River. MEF cells wereexpanded in DMEM medium supplemented with 10% FBS (Hyclone), 2 mMglutamine, and 100 mM MEM nonessential amino acids. Sub-confluent MEFcell cultures were treated with 10 μg/ml mitomycin C (Sigma, St. Louis,MO) for 3 h to arrest cell division, then trypsinized and plated at2×104/cm2 on 0.1% bovine gelatin-coated dishes. MEF cells from passagetwo through four were used as feeder layers. Human embryonic stem cellsplated on MEF cell feeder layers were cultured at 37° C. in anatmosphere of 5% CO₂ within a humidified tissue culture incubator. Whenconfluent (approximately 5 to 7 days after plating), human embryonicstem cells were treated with 1 mg/ml collagenase type IV(Invitrogen/GIBCO) for 5 to 10 min and then gently scraped off thesurface using a 5 ml glass pipette. Cells were centrifuged at 900 rpmfor 5 min, and the pellet was resuspended and re-plated at a 1:3 to 1:4ratio of cells on plates coated with a 1:30 dilution of growth factorreduced MATRIGEL™ (BD Biosciences). Cells were subsequently cultured inMEF-conditioned media supplemented with 8 ng/ml bFGF and collagenasepassaged on MATRIGEL coated plates for at least five passages. The cellscultured on MATRIGEL™ were routinely passaged with collagenase IV(Invitrogen/GIBCO), Dispase (BD Biosciences) or Liberase enzyme (Roche,IN).

Example 14 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with Extracellular Matrix to DefinitiveEndoderm

Differentiation of embryonic stem cells to definitive endoderm wascarried out as previously described in Nature Biotechnology 23,1534-1541 (December 2005). Briefly, H9 cultures at approximately 60 to70% confluency were exposed to DMEM:/F12 medium supplemented with 0.5%FBS and 100 ng/ml activin A for two days, followed by treatment withDMEM/F12 medium supplemented with 2% FBS and 100 ng/ml activin A (AA)for an additional three days. H9 cells were cultured on plates coatedwith growth factor reduced MATRIGEL at a 1:30 to 1:10 dilution or onregular MATRIGEL at al:30 to 1:10 dilution The plates were coated withMATRIGEL for 1 hr at room temperature.

At day 5, the cultures were analyzed by FACS for CXCR4, E-cadherin, CD9,and N-cadherin expression and by real time PCR for SOX-17, SOX-7,Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC),HNF-3 beta, and GATA4. AFP and SOX-7 are regarded as visceral endodermmarkers, while GATA4, HNF-3 beta and SOX-17 represent definite endodermmarkers, and GSC, Bry, and CXCR4 represent markers of primitive streak.FIGS. 17A, 17B, 17C, 17D, 17E and 17F depicts the expression of CXCR4 byFACS. There was a significant increase in expression of CXCR4 by cellscultured on plates coated with MATRIGEL at a 1:10 dilution as comparedto lower concentrations of MATRIGEL. Furthermore, growth factor reducedMATRIGEL was not as effective in formation of definitive endoderm cellsas compared to regular MATRIGEL.

FIG. 18 shows the real-time PCR results verifying that cells cultured onplates coated with a 1:10 dilution of MATRIGEL showed a significantupregulation of definitive endoderm markers as compared to cellscultured on a 1:30 dilution of MATRIGEL.

Example 15 Microarray Analysis of Changes in Gene Expression inEmbryonic Stem Cells Following Formation of Definitive Endoderm

Total RNA was isolated from the following human embryonic stem cellcultures using an RNeasy mini kit (Qiagen): H9P83 cells cultured onMATRIGEL-coated plates and exposed to DMEM/F12 medium supplemented with0.5% FBS and 100 ng/ml activin A for two days followed by treatment withDMEM/F12 medium supplemented with 2% FBS and 100 ng/ml Activin A (AA)for an additional three days; H9P44 cells cultured on MEFs and exposedto DMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin Afor two days followed by treatment with DMEM/F12 medium supplementedwith 2% FBS and 100 ng/ml activin A for an additional three days.Controls for each group included cells plated on MATRIGEL-coated dishesand cultured in MEF-conditioned medium or cells plated on MEFs andcultured in ES medium.

Sample preparation, hybridization, and image analysis were performedaccording to the Affymetrix Human Genome U133 Plus 2.0 Array. Followingnormalization and a log transformation, data analysis was performedusing OmniViz® software (MA) and GENESIFTER (VizXLabs, WA). Thevariability within each treatment and among the different treatments wascompared using the Pearson correlation coefficient. Variance in geneexpression profiles between the different treatments along with thecorrelation coefficient between the lines are depicted in FIGS. 19A and19B. Significant differences in gene expression between the samples wereevaluated using analysis of variance and an F-test with adjusted P-value(Benjamini and Hochberg correction) of ≤0.05. Only genes with a presentcall were included in the analysis. Table IV lists the genes that aredifferentially expressed with a difference at least 5-fold between thevarious samples. The normalized intensity value of the genes that aresignificantly expressed along with the standard error of the mean (SEM)for each gene are listed.

Example 16 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm

Differentiation of embryonic stem cells to definitive endoderm wascarried out as previously described in Nature Biotechnology 23,1534-1541 (December 2005). Briefly, H9, H7, or H1 cells seeded on growthfactor reduced MATRIGEL™ (1:30 dilution) cultures at approximately 60 to70% confluency were exposed to DMEM/F12 medium supplemented with 0.5%FBS and 100 ng/ml activin A (R&D Systems, MN)) for two days followed bytreatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/mlactivin A (AA) for an additional three days. In all subsequent examplesunless otherwise noted, this treatment regimen will be referred to asthe definite endoderm (DE) protocol. In parallel, H9, H7, or H1 cellscultured on MEF feeders were also exposed to the same DE protocoloutlined above.

At day 5, the cultures were analyzed by FACS for CXCR4, E-cadherin, CD9,CD99, and N-cadherin (CD56) expression and by real time PCR for SOX-17,SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid(GSC), HNF-3 beta, and GATA4. AFP and SOX-7 are regarded as visceralendoderm markers while GATA4, HNF-3beta and SOX-17 represent definiteendoderm markers and GSC, Bry, and CXCR4 represent markers of primitivestreak.

H-lines cultured on mouse feeders and exposed to the DE protocolresulted in a robust expression of DE markers and expression of CXCR4 byFACS (FIGS. 20A, 20B and 20C). There was also a significant decrease inexpression of E-cadherin following treatment with the DE protocol.Lastly, the CXCR4+ population also stained positive for CD117. FIGS. 21Aand 21B shows a significant upregulation of definitive endoderm markersas compared to untreated H7 (FIG. 21A) and H9 cells (FIG. 21B).

Unlike H-lines cultured on MEF feeders, H-lines cultured on MATRIGEL™(1:30 dilution) and treated with the definitive endoderm protocol failedto show robust expression of definitive endoderm markers. In particular,the expression of CXCR4 by FACS and by real-time PCR was significantlylower for cells cultured on MATRIGEL™ as compared to cells cultured onmouse embryonic fibroblasts. Expression of definitive endoderm markersfollows a general response pattern with H1 being greater than H9, whichis greater than H7 (FIGS. 22A, 22B, 22C, 23A, 23B and 23C). From FIGS.22A, 22B and 22C, H1 cells showed a significant increase in CXCR4expression as compared to H7 and H9 lines. Note that in all cases, theexpression of CXCR4 was lower for cells cultured on MATRIGEL™ (1:30dilution) as compared to cells cultured on mouse embryonic fibroblasts.FIGS. 23A, 23B, and 23C shows the real-time PCR results showing thatthere was modest increase in upregulation of definitive endoderm markersin H7 (FIG. 23A) and H9 (FIG. 23B) lines. However, H1 (FIG. 23C) lineshowed a more robust upregulation of definitive endoderm markers ascompared to H7 and H9 lines.

Example 17 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm(DE)-Role of Wnt Ligands

H7P44 and H9P46 embryonic stem cells were cultured on MATRIGEL™ (1:10dilution) coated dishes and exposed to DMEM/F12 medium supplemented with0.5% FBS, and 100 ng/ml activin A (R&D Systems, MN) for two daysfollowed by treatment with DMEM/F12 media supplemented with 2% FBS and100 ng/ml activin A (AA) for an additional three days. In some of thecultures 20 ng/ml Wnt-3a (Catalog #1324-WN-002, R&D Systems, MN), 20ng/ml Wnt-5a (Catalog #654-WN-010, R&D Systems, MN), 25 ng/ml Wnt-7a(Catalog #3008-WN-025, R&D Systems, MN), or 25 ng/ml Wnt-5b (Catalog#3006-WN-025, R&D Systems, MN) was added throughout the five daytreatment. FIGS. 24A and 24B depicts phase contrast images of H9P46definitive endoderm culture in the presence of high concentration of (a)AA or (b) AA+20 ng/ml Wnt-3a. FIGS. 25A, 25B, 25C and 25D depicts theexpression of CXCR4 by FACS at day 5 for H7P44, and H9P46 lines culturedon MATRIGEL™ (1:30 dilution) and exposed to the DE protocol+Wnt-3a(FIGS. 25B and 25D) and −Wnt-3a (FIGS. 25A and C). Presence of Wnt-3a inDE cultures resulted in robust expression of CXCR4 (CD184) as comparedto DE cultures treated with low serum plus high concentration of AA.FIGS. 26A and 26B displays the real-time PCR data for a) H7 and b) H9cultures treated with low serum+AA+/−Wnt ligands. For both H-lines,addition of WNT-3a resulted in significant upregulation of definitiveendoderm markers. In contrast, Wnt 5a, Wnt-5b and Wnt-7a had minimalimpact on expression of definitive endoderm markers.

Example 18 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm:Effective Dose of Wnt-3a

H9P46 embryonic stem cells were cultured on MATRIGEL™ coated dishes(1:10 dilution) and exposed to DMEM/F12 medium supplemented with 0.5%FBS, 100 ng/ml Activin A (AA), and 10-50 ng/ml WNt-3a (R&D Systems, MN)for two days followed by treatment with DMEM/F12 media supplemented with2% FBS, 100 ng/ml activin A (AA), and 10-50 ng/ml Wnt-3a for anadditional three days. Control cultures were not treated with Wnt-3a.FIG. 27A depicts the expression of CXCR4 by FACS at day 5 in the absenceof Wnt-3a, b) 10 ng/ml Wnt-3a, c) 20 ng/ml Wnt-3a and d) 50 ng/mlWnt-3a. In the absence of Wnt-3a the expression of CXCR4 was very low.In contrast, addition of 10-50 ng/ml of Wnt-3a significantly increasedthe number of CXCR4 positive cells. Furthermore, addition of 10 ng/ml ofWnt-3a was as effective as addition of 50 ng/ml of Wnt-3a. Real-time PCRresults (FIG. 28A) also confirm this finding.

In a separate study, H9p52 cells were plated on 1:30 low growth factorMATRIGEL™. For the first 2 days of the DE protocol a range of Wnt3Adoses was used: 10 ng/ml, 5 ng/ml and 1 ng/ml. FIG. 28B shows PCRanalysis of the DE markers after 5 days of treatment. The number ofcells at the end of the experiment is noted in FIG. 28C. This indicatesthat cells are proliferating when higher doses of Wnt-3a are used.Extension to 5 days of Wnt3a treatment (5D) had little effect on DEmarkers by PCR and did not significantly increase cell numbers (FIG.28C). These data indicate that 10 ng/ml Wnt3a for 2 days is sufficientto reach optimal cell expansion and definitive endoderm differentiation.

Example 19 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm(DE)-Effect of GSK-3B Inhibitor

In order to confirm that the effect of Wnt-3a was through the Wntpathway, a GSK-3 inhibitor was used to activate the downstream targetsof Wnt, such as beta catenin. H9P46-P48 embryonic stem cells werecultured on MATRIGEL™ coated dishes (1:10 dilution) and exposed toDMEM/F12 medium supplemented with 0.5% FBS, 100 ng/ml activin-A (AA),and 10-1000 nM GSK-3B inhibitor IX (Catalog #361550, Calbiochem, CA) fortwo days followed by treatment with DMEM/F12 media supplemented with 2%FBS, 100 ng/ml activin A (AA), and 0-1000 nM GSK-3B inhibitor IX(Catalog #361550, Calbiochem, CA) for an additional three days. Controlcultures were treated with low serum plus high dose of activinA+/−Wnt-3a. FIG. 29A depicts the expression of CXCR4 by FACS at day 5 inthe absence of Wnt-3a or GSK-3B inhibitor, b)+20 ng/ml Wnt-3a, c)+1000nM GSK-3B inhibitor IX, d)+500 nM GSK-3B inhibitor IX, e)+100 nM GSK-3Binhibitor IX, f)+10 nM GSK-3B inhibitor IX, g)+100 nM GSK-3B inhibitorIX for days 1-2, and h)+10 nM GSK-3B inhibitor IX for days 1-2.

In the absence of Wnt-3a or at 10 nm GSK-3B inhibitor the expression ofCXCR4 was very low. In contrast, addition of 20 ng/ml of Wnt-3a or100-1000 nM GSK-3B inhibitor significantly increased the number of CXCR4positive cells. Furthermore, addition of 100 nM GSK-3B inhibitor fordays 1-2 was as effective as addition of 100 nM GSK-3B inhibitor for theentire five-day period. FIGS. 30A and 30B depicts the gene expression ofdefinitive endoderm markers for (FIG. 30A) H9P48 cells and (FIG. 30B)H9P46 cells.

FIG. 16 depicts the outline of a differentiation protocol in thisinvention, where embryonic stem cells are differentiated into definitiveendoderm in a feeder free system.

Example 20 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm(DE)-Effective Dose of Activin-A in the Presence of GSK-3B Inhibitor orWnt-3A

H9P49 and H1P46 embryonic stem cells were cultured on MATRIGEL™ coateddishes (1:10 dilution) and exposed to DMEM/F12 medium supplemented with0.5% FBS, 10-100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX(Catalog #361550, Calbiochem, CA) or 20 ng/ml Wnt-3a for two daysfollowed by treatment with DMEM/F12 media supplemented with 2% FBS,10-100 ng/ml activin A (AA) for an additional three days. Controlcultures were treated with low serum plus 100 ng/ml of activin A. FIGS.31A, 31B, 31C, 31D, 31E and 31F depicts the expression of CXCR4 by FACSfor H9P49 and H1P46 at day 5 with a) 10 ng/ml activin A for all fivedays plus 20 ng/ml of Wnt-3A for the first two days, b) 100 ng/mlactivin A for all five days plus 20 ng/ml of Wnt-3A for the first twodays c) 100 ng/ml activin A for all five days plus 100 nM of GSK-3Binhibitor IX for the first two days d) 10 ng/ml activin A for all fivedays plus 100 nM of GSK-3B inhibitor IX for the first two days, e) 100ng/ml activin A for all five days plus ng/ml of Wnt-3A for the first twodays, and f) 10 ng/ml activin A for all five days plus 20 ng/ml ofWnt-3A for the first two days. FIGS. 31A, 31B, 31C and 31D is for H9P49cells and FIGS. 31E and 31F is for H1P46 cells. FIGS. 32A and 32Bdepicts the gene expression of definitive endoderm markers for H9P49cultures treated with 10, 50, or 100 ng/ml of activin A plus ng/ml ofWnt-3a: FIG. 32A: expression of AFP, Bry, CXCR4, GSC, HNF-3B, and POU5F(Oct-4) and FIG. 32B: SOX-17 and GATA4. This does not add up- It appearsto be a problem with figure numbers or it is a cut and paste problem. Itappears that robust expression of definitive endoderm markers can beobtained by using 50 ng/ml of AA+20 ng/ml of Wnt-3A or 100 nM GSK-3Binhibitor IX. Lower doses of activin A lead to formation ofextraembryonic endoderm.

Example 21 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm(DE)-Combination of Wnt-3a and GSK-3B Inhibitor

H9P53 embryonic stem cells were cultured on MATRIGEL™ coated dishes(1:30 dilution) and exposed to DMEM/F12 medium supplemented with 0.5%FBS, 100 ng/ml activin A (AA), and 100 nM GSK-3B inhibitor IX (Catalog#361550, Calbiochem, CA)+/−20 ng/ml Wnt-3a for two days followed bytreatment with DMEM/F12 media supplemented with 2% FBS, 10-100 ng/mlactivin-A (AA) for an additional three days. In parallel, H9P53 cultureswere treated with 25 ng/ml BMP-4 (Catalog #314-BP-010, R&D Systems,MN)+/−ng/ml Wnt-3A+/−100 ng/ml activin A. Control cultures were treatedwith low serum plus 100 ng/ml of activin A. FIGS. 33A, 33B, 33C, 33D,33E, and 33F depicts the expression of CXCR4 by FACS at day 5 with a)100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A for thefirst two days and 25 ng/ml BMP-4 for days 3-5, b) 100 ng/ml activin Afor all five days plus 20 ng/ml of Wnt-3A for the first two days c) 100ng/ml activin A for all five days plus 100 nM of GSK-3B inhibitor IX forthe first two days d) 20 ng/ml Wnt-3a+25 ng/ml BMP-4 for all five days,e) 100 ng/ml activin A for all five days plus 20 ng/ml of Wnt-3A+100 nmGSK-3B inhibitor IX for the first two days, and f) 100 ng/ml activinA+25 ng/ml BMP-4 for all five days. FIGS. 34A and 34B depicts the geneexpression of definitive endoderm markers, as determined by real-timePCR for cultures of the human embryonic stem cell line H1 at passage 46,treated with 10 or 100 ng/ml of activin A plus ng/ml of Wnt-3a or 100 NMGSK-3B inhibitor: panel (a): expression of AFP, Bry, CXCR4, GSC, andPOU5F (Oct-4) and panel (b): SOX-17, HNF-3B, and GATA4. Results areexpressed as fold increase over untreated cells. FIGS. 35A and 35Bdepicts the gene expression of definitive endoderm markers, asdetermined by real-time PCR for cultures of the human embryonic stemcell line H9 at passage 49, treated with 50 or 100 ng/ml of activin Aplus or 100 nM GSK-3B inhibitor: panel (a): expression of AFP, Bry,CXCR4, GSC, HNF-3B, and POU5F (Oct-4) and panel (b): SOX-17 and GATA4.Results are expressed as fold increase over untreated cells. FIGS. 36Aand 36B depicts the gene expression of definitive endoderm markers forH9P53 culture treated with combinations of activin A, Wnt-3a, GSK-3inhibitor, and BMP-4: a) expression of AFP, Bry, CXCR4, GSC, HNF-3B, andSOX7 and b) SOX-17, HNF-3B and GATA4. Addition of BMP-4 to the DEprotocol appears to induce formation of mesoderm marker BRY andcombination of Wnt-3A and GSK-4B inhibitor do not lead to significantupregulation of definitive endoderm markers as compared to addition ofeach agent by itself in the presence of activin A.

Example 22 Differentiation of Human Embryonic Stem Cells Cultured onMEFs to Definitive Endoderm (DE)-Combination of Wnt-3a, Activin A,Wnt-5a, BMP-2, BMP-4, BMP-6, BMP-7, IL-4, and SDF-1 in Low Serum

H9P44 cells were plated onto 6 well plates previously coated withmitomycin treated mouse embryonic fibroblasts (MEF). Cells were grownuntil 70 to 80% confluency in ES cell medium consisting of DMEM/F12(Invitrogen/GIBCO) supplemented with 20% knockout serum replacement, 100nM MEM nonessential amino acids, 0.5 mM beta-mercaptoethanol, 2 mML-glutamine (all from Invitrogen/GIBCO) and 8 ng/ml human basicfibroblast growth factor (bFGF) (R&D Systems).

For DE formation, cells were treated in the presence or absence ofActivin A (100 ng/ml) in addition to other growth factors detailedbelow. Growth factors were added to increasing concentration of FBS in astepwise manner as indicated in the following regimen:

-   -   Day 0: 0% FBS in DMEM/F12    -   Day 1: 0.5% FBS in DMEM/F12    -   Day 2: 2% FBS in DMEM/F12.    -   Day 3: Cells were harvested for FACS analysis and RT-PCR.

All growth factors were purchased from R&D Systems, MN. A detaileddescription and concentration of growth factors for each treatment groupis shown below.

-   -   1. Control—No growth factor added    -   2. Activin A (100 ng/ml)    -   3. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt5a (10 ng/ml)    -   4. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt5a (10 ng/ml)+BMP2        (100 ng/ml)    -   5. Activin A (100 ng/ml)+BMP-4 (100 ng/ml)    -   6. Activin A (100 ng/ml)+BMP-6 (100 ng/ml)    -   7. Activin A (100 ng/ml)+BMP-7 (100 ng/ml)    -   8. Activin A (100 ng/ml)+BMP-4 (100 ng/ml)+BMP-6 (100        ng/ml)+BMP-7 (100 ng/ml)    -   9. IL-4 (10 ng/ml)    -   10. SDF1a (20 ng/ml)    -   11. Activin A (100 ng/ml)+IL-4 (10 ng/ml)+SDF1a (20 ng/ml)    -   12. BMP2 (100 ng/ml)+BMP-4 (100 ng·ml)+BMP-6 (100 ng/ml)+BMP-7        (100 ng/ml)    -   13. Activin A (100 ng/ml) BMP-2 (100 ng/ml)+BMP-4 (100        ng·ml)+BMP-6 (100 ng/ml)+BMP-7 (100 ng/ml)    -   14. Activin A (100 ng/ml)+IL-4 (10 ng/ml)    -   15. Activin A (100 ng/ml)+(SDF1a (20 ng/ml)    -   16. Activin A (100 ng/ml)+Wnt-3a (10 ng/ml)+Wnt-5a (10        ng/ml)+Wnt-7a (10 ng/ml)    -   17. Activin A (100 ng/ml)+IL-4 (10 ng/ml)+SDF1a (20 ng/ml)+BMP-4        (100 ng/ml)

Results:

Cells were harvested on Day 3 of DE protocol treatment. For analysis, analiquot of treated cells was used for RNA preparation for RT-PCR and therest of cells used for FACS analysis. The frequency (%) of CXCR4 isshown in FIG. 37 . Addition of the above growth factor(s) did notenhance expression of CXCR4 above treatment with 100 ng/ml AA in lowserum.

For RT-PCR analysis, cells were analyzed for expression of selectedpanel of definitive endoderm markers. Results shown were calibratedagainst cells grown in the base medium but not treated with Activin A orany of the other growth factors. In agreement with the FACS data, TableV shows that there was no significant upregulation of definitiveendoderm markers by addition of growth factors, such as Wnt-3a tocultures treated with a high dose of activin A in low serum. This is incontrast to the previous examples showing a significant increase in DEmarkers for ES cells cultured on feeder-free conditions in the presenceof activin A, WNT3A, and low serum.

Example 23 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ or Human Fibronectin toDefinitive Endoderm (DE)

H9P55 cells were grown and differentiated on human fibronectin orregular growth factor MATRIGEL™ (BD Biosciences). 1 ml of DMEM/F12(Invitrogen/GIBCO) containing 1 ug/ml of human fibronectin (R&D systems,MN) was added to each well of 6 well tissue culture treated dish.Alternatively, regular growth factor MATRIGEL™ was diluted 1:10 inDMEM/F12 and 1 ml of diluted MATRIGEL™ was added to each well of 6 welltissue culture treated dish. Cells were passed with collagenase. Aftercells reached 80% confluency, there were treated as follows: 2 days 0.5%FBS containing 10 ng/ml mouse recombinant Wnt3a (R&D) and 100 ng/mlActivin A (R&D). This was followed by 3 days 2% FBS plus 100 ng/mlActivin A. FIGS. 38A and 38B depict the expression of CXCR4 by embryonicstem cells cultured on fibronectin and MATRIGEL, respectively. Real-timePCR results (FIG. 39 ) confirm that formation of definitive endoderm wasequivalent on fibronectin and MATRIGEL™ coated plates.

Example 24 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with Varying Concentrations of MATRIGEL™to Definitive Endoderm

H9 cultures at approximately 60 to 70% confluency were exposed toDMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt-3a and 100ng/ml activin A for two days followed by treatment with DMEM/F12 mediasupplemented with 2% FBS, 20 ng/ml Wnt-3a and 100 ng/ml activin A (AA)for an additional three days. H9 cells were cultured on plates coatedwith regular MATRIGEL at a 1:60 to 1:10 dilution. The plates were coatedwith MATRIGEL for 1 hr at room temperature.

Real time PCR results are shown in FIG. 40 . Treatment of humanembryonic stem cells with low serum, Activin A and Wnt3a led to theexpression of CXCR4, GATA4, Goosecoid, HNF-3beta, and SOX-17 genes,suggesting that the cells were differentiating to the definitiveendoderm stage. However, it does not appear that the in the presence ofWnt-3A concentration of the MATRIGEL™ coating plays an important role indifferentiation.

Example 25 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with Extracellular Matrix andSubsequently Cultured on MEFS and Differentiated to DefinitiveEndoderm-Role of Wnt-3a

Cells from the human embryonic stem cell line H9 cultured on MATRIGEL™for at least five passages were seeded onto MEF feeders in ES media.When the cells reached 60 to 70% confluency they were exposed toDMEM/F12 medium supplemented with 0.5% FBS and 100 ng/ml activin A fortwo days followed by treatment with DMEM/F12 media supplemented with 2%FBS and 100 ng/ml activin A (AA) for an additional three days.Additional treatment groups include Wnt-3a at 20 ng/ml for all fivedays+10-100 ng/ml of activin A.

At day 3 and 5, the cultures were analyzed by real time PCR for SOX-17,SOX-7, Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid(GSC), HNF-3 beta, GATA4, hTERT and Oct4. AFP and SOX-7 are regarded asvisceral endoderm markers while GATA4, HNF-3beta and SOX-17 representdefinite endoderm markers and GSC, Bry, and CXCR4 represent markers ofprimitive streak. hTERT and Oct-4 are markers for self renewal andpluripotency respectively. Real time-PCR results are shown in FIGS. 41A,41B, 41C and 41D. FACS analysis was also performed at day 3 and 5.Expression levels of CXCR-4, and CD9 were analyzed and reported in FIG.41E.

In the absence of Wnt3a, AFP expression levels of cells cultured in 100ng/ml Activin A are similar to those seen in untreated controls.However, with the addition of Wnt3a to cells cultured in 100 ng/mlactivin A, there is an increase in the expression of AFP that increasesover time. When a lower concentration of Activin A is used, AFPexpression is very high, regardless of the presence of Wnt3a (FIG. 41A).This suggests that a high concentration of Activin A is necessary tokeep the cells from differentiating to extra-embryonic tissues.

By FACS analysis, CXCR4 positive cells ranged from 32-42% of thepopulation in samples treated with a high concentration of Activin A butnot treated with Wnt3a as compared to 23-33% of the population insamples treated with a high concentration of Activin A and Wnt3a at day3 (FIG. 41E). By day 5 of treatment, 28-32% of cells treated with a highconcentration of activin A but not treated with Wnt-3a expressed CXCR4as compared to 43-51% of cells treated with a high concentration ofActivin A and Wnt3a (FIG. 41F). In cells treated with a lowconcentration of Activin A, there were more CXCR4 positive cells in thetreatment group without Wnt3a (11 to 20%) as compared to the Wnt-3atreated group (3 to 4%) (FIG. 41G). Overall, Wnt-3a does not appear toplay a significant role in differentiation of human embryonic stemcells, cultured on MEFs, to definitive endoderm. This suggests that thefeeder layer is probably secreting sufficient Wnt-3a or analogous ligandto enhance activin A induced definitive endoderm formation.

Example 26 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with Extracellular Matrix to DefinitiveEndoderm Following Treatment with a Wnt Inhibitor-DKK-1

To determine if the addition of Wnt-3a was causing the increase indifferentiation, an inhibitor of Wnt-3 signaling was added to thecultures. H9 cultures at approximately 60 to 70% confluency were exposedto DMEM/F12 medium supplemented with 0.5% FBS, 20 ng/ml Wnt3a, 100 ng/mlDikkopf-1 (DKK-1) and 100 ng/ml activin A for two days followed bytreatment with DMEM/F12 media supplemented with 2% FBS and 100 ng/mlactivin A (AA) for an additional three days. H9 cells were cultured onplates coated with Growth Factor Reduced MATRIGEL at a 1:30 dilution.The plates were coated with MATRIGEL for 1 hr at room temperature.

At day 5, the cultures were analyzed by real time PCR for SOX-17, SOX-7,Alpha-fetal protein (AFP), CXCR4, Brychyury (Bry), gooscecoid (GSC),HNF-3 beta, GATA4, hTERT and Oct4. AFP and SOX-7 are regarded asvisceral endoderm markers while GATA4, HNF-3beta and SOX-17 representdefinite endoderm markers and GSC, Bry, and CXCR4 represent markers ofprimitive streak. hTERT and Oct-4 are markers for self renewal andpluripotency respectively. Results are shown in FIG. 42 .

In the presence of Wnt3a, cells express CXCR4, GATA4, HNF-3beta andSOX17, all markers of definitive endoderm. Markers of primitive streakformation such as goosecoid were also detected at levels higher thanthat detected in untreated controls. With the addition of DKK1, theexpression level of the aforementioned differentiation markersdramatically decrease to levels similar to that of untreated cells.

Example 27 Immunofluorescence Staining of DE Markers for H9 EmbryonicStem Cells Cultured on Tissue Culture Substrate Coated MATRIGEL andDifferentiated in Low Serum Plus Activin-a and +/−Wnt-3a

Day 5 DE cultures of H9 cells were stained according to Example 10 forSOX-17, HNF-3B, GATA-4, N-cadherin, and E-cadherin. All nuclei werecounter stained with DAPI. 20 ng/ml Wnt-3a resulted in significantlylarger number of nuclei stained positive for SOX-17, HNF-3beta. andGATA-4 as compared to cultures differentiated in the absence of Wnt-3a.Furthermore, addition of Wnt-3a resulted in significant loss ofexpression of e-cadherin and enhanced expression of N-cadherin (FIGS.43A and 43B).

Example 28 Microarray Analysis of Changes in Gene Expression inEmbryonic Stem Cells Following Formation of Definitive Endoderm on MEFsVs MATRIGEL™

Total RNA was isolated from the following embryonic stem cell culturesusing an RNeasy mini kit (Qiagen): A) H9P33 cells cultured onMATRIGEL™-coated plates (1:30 dilution) and exposed to DMEM/F12 mediumsupplemented with 0.5% FBS and 100 ng/ml activin A for two days followedby treatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/mlactivin A (AA) for an additional three days; B) H9P44 cells cultured onMEFs and exposed to DMEM/F12 medium supplemented with 0.5% FBS and 100ng/ml Activin A for two days followed by treatment with DMEM/F12 mediumsupplemented with 2% FBS and 100 ng/ml Activin A for an additional threedays, and C) H9P48 cells cultured on MATRIGEL™-coated plates (1:30dilution) and exposed to DMEM/F12 medium supplemented with 0.5% FBS and100 ng/ml activin A plus 20 ng/ml Wnt-3A for two days followed bytreatment with DMEM/F12 medium supplemented with 2% FBS and 100 ng/mlActivin A (AA) for an additional three days. Controls for each groupincluded cells plated on MATRIGEL-coated dishes and cultured inMEF-conditioned medium or cells plated on MEFs and cultured in ESmedium. All groups contained three biological replicates and eachbiological replicate was repeated on two separate gene chips.

Sample preparation, hybridization, and image analysis were performedaccording to the Affymetrix Human Genome U133 Plus 2.0 Array. Followingnormalization and a log transformation, data analysis was performedusing OmniViz® software (MA) and GENESIFTER (VizXLabs, WA). Significantdifferences in gene expression between the samples were evaluated usinganalysis of variance and an F-test with adjusted P-value (Benjamini andHochberg correction) of ≤0.05. Only genes with a present call in atleast one group were included in the analysis. Table VI lists the meannormalized log-transformed signal intensity of genes showing at least5-fold difference between group A, group B, and group C along with theadjusted P-value for each gene.

Example 29 Differentiation of SA002 ES Line Cultured on Tissue CultureSubstrate Coated with MATRIGEL™ to Definitive Endoderm (DE)

SA002 P38 cells (Cellartis, Sweden) previously cultured for at leastthree passages on MATRIGEL-coated plates (1:30 dilution) in MEF-CMsupplemented with 8 ng/ml of bFGF were exposed to DMEM/F12 mediumsupplemented with 0.5% FBS, and 100 ng/ml activin A (R&D Systems,MN)+/−20 ng/ml of Wnt-3a or 100 nm GSK-3B IX inhibitor for two daysfollowed by treatment with DMEM/F12 media supplemented with 2% FBS and100 ng/ml activin A (AA) for an additional three days. Real time PCRresults are shown in FIGS. 44A and 44B. Similar to H1, H7, and H9 lines,SA002 line also required addition of Wnt-3A for robust expression of DEmarkers. Expression of CXCR4 is depicted in FIGS. 45A, 45B and 45C: a)AA treatment b) AA+Wnt-3a c) AA+GSK-3B inhibitor.

Example 30 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with Human Serum to Definitive Endoderm(DE)

Cultures of the human embryonic stem cell line H1 at passage 55 weregrown and differentiated on human serum (Sigma, #H1388, MO) coatedplates. 0.5 ml of human serum was added to each well of 6 well tissueculture treated dish, incubated for 1 hr at room temperature, andaspirated before adding human embryonic stem cells. After cells reached80% confluency, they were treated as follows: 2 days 0.5% FBS containing10 ng/ml mouse recombinant Wnt3a (R&D) or 100 nM GSK-3B inhibitor IX(Catalog #361550, Calbiochem, CA) and 100 ng/ml Activin A (R&D). Thiswas followed by 3 days 2% FBS plus 100 ng/ml Activin A. Cultures werethen analyzed by real-time PCR (FIGS. 46A and 46B). Robust expression ofdefinitive endoderm markers were noted for cells treated with activinA+GSK-3B inhibitor or Wnt-3A as compared to cells treated with activin Aonly. These findings parallel our findings for human embryonic stemcells cultured on MATRIGEL™ or human fibronectin coated plates.

Example 31 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm(DE)-Evaluation of Various GSK-3B Inhibitor

The effectiveness of a number of commercially available GSK-3Binhibitors was evaluated in formation of DE from human embryonic stemcells. The following GSK-3B inhibitors were evaluated at 100 nM: GSK-3Binhibitor VIII (Catalog #361549, Calbiochem, CA), GSK-3B inhibitor IX(Catalog #361550, Calbiochem, CA), GSK-3B inhibitor XI (Catalog #361553,Calbiochem, CA), GSK-3B inhibitor XII (Catalog #361554, Calbiochem, CA).H1P54 ES cells were cultured on MATRIGEL™ coated dishes (1:30 dilution)and exposed to DMEM/F12 medium supplemented with 0.5% FBS, 100 ng/mlactivin A (AA)+/−various GSK-3B inhibitors for two days followed bytreatment with DMEM/F12 media supplemented with 2% FBS, 100 ng/mlactivin A (AA) for an additional three days. Control cultures weretreated with low serum plus high dose of AA. FIGS. 47A and 47B depictsthe gene expression of definitive endoderm markers at day 5. GSK-3Binhibitor IX and XI were both effective in inducing DE formation ascompared to GSK-3B inhibitor VIII and XII.

Example 32 Formation of Pancreatic Endoderm by Human Embryonic StemCells Cultured Under Feeder-Free Conditions: Evaluation of Retinoic AcidAnalogues

H9P49 embryonic stem cells were cultured on MATRIGEL™ (1:30 dilution)coated dishes and exposed to DMEM/F12 medium supplemented with 0.5% FBS,20 ng/ml Wnt-3a (Catalog #1324-WN-002, R&D Systems, MN), and 100 ng/mlactivin A (R&D Systems, MN) for two days followed by treatment withDMEM/F12 media supplemented with 2% FBS and 100 ng/ml activin A (AA) foran additional three days. At day 5, cells were collected for evaluationby FACS and real-time PCR. As indicated in previous examples, thisprotocol resulted in robust upregulation of definitive endoderm markers,such as CXCR4 and SOX-17. The resulting definitive endoderm cells at day5 were exposed to the following media conditions to induce pancreaticendoderm formation: culturing in DMEM/F12 media supplemented with 2% FBSand 1 μM all-trans retinoic acid (RA) (Catalog #R2625, Sigma, MO), or0.1-10 μM AM-580(4-[(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)carboxamido]benzoicacid, Catalog #A8843, Sigma, MO), or 0.1-1 μM TTNPB(4-[(E)-2-(5,6,7,8-Tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl]benzoicacid Arotinoid acid, Catalog #T3757, Sigma, MO) for 3 days. AM-580 andTTNPB are retinoic acid analogs with affinity for retinoic acidreceptors. RA treatment was followed by additional three day treatmentin DMEM/F12 media supplemented with 2% FBS and 20-ng/ml bFGF (Catalog#F0291, Sigma, MO). Cultures were harvested and samples of mRNA werecollected for analysis.

Gene expression analysis revealed that (FIGS. 48A, 48B, 48C and 48D)addition of 1 μM RA followed by exposure to bFGF significantlyupregulates pancreatic endoderm markers, such as PDX-1. Furthermore,this protocol resulted in robust expression of foregut endoderm markers,such as CDX-2 and AFP. At 1 μM concentration, addition of RA analogsresulted in equivalent pancreatic endoderm and foregut markers. However,addition of 1 μM RA analogs resulted in more robust expression of AFP ascompared to all-trans retinoic acid. However, addition of 10 μM AM-580suppressed AFP and CDX-2 expression while maintaining a high expressionof PDX-1.

Example 34 The Effect of Wnt-3a Treatment on Cytokine Expression inHuman Embryonic Stem Cells

The effect that Wnt-3a treatment has on cytokine expression was analyzedusing a protein array. Cells of the human embryonic stem cell line H9were cultured according to the methods described in Example 15. Atpassage 54, cells were differentiated in the presence of 100 ng/mlActivinA+/−10 ng/ml Wnt3a for 2 days in 0.5% FBS DMEM/F12. Cells weresubsequently cultured for an additional three days in 100 ng/ml ActivinA and 2% FBS DMEM/F12. At the end of the 5th day, CXCR4 expression wasdetermined by FACS for each treatment group. Cells treated with ActivinA only had 1% of cells expressing CXCR4. Cells treated with Activin Aand Wnt3a had 73% of cells positive for CXCR4 expression.

Cell lysates were prepared from cells of each treatment group, with amammalian cell lysis kit (Sigma-Aldrich, MO). Conditioned media fromeach treatment group was collected and concentrated. Cytokine arrayanalysis was completed using Cytokine Array panels provided byRayBiotech, GA (http://www.raybiotech.com/). Table VII lists cytokine,growth factor, and receptor expression following normalization of thedata and background subtraction. For each panel, positive and negativecontrols are also included. Th data shown are two independent samplesper cell treatment group (1,2).

Noticeable upregulation of Angiogenin, IGFBP-1 and EGF are seen in theWnt3 treated cell conditioned media. Numerous proteins are upregulatedin the Wnt3a treated cell lysates including IGFBP-1, TGFbeta-1 andTGFbeta-3. These upregulated proteins can be added back into thedifferentiation media to replace or enhance Wnt3a effects on definitiveendoderm formation.

Example 35 Differentiation of Human Embryonic Stem Cells Cultured onTissue Culture Substrate Coated with MATRIGEL™ to Definitive Endoderm:Role of Wnt1

H1P55 ES cells were cultured on MATRIGEL™ (1:30 dilution) coated dishesand exposed to DMEM/F12 medium supplemented with 0.5% FBS, and 100 ng/mlactivin A+/−10-20 ng/ml of WNT-1 (PeproTech, NJ, Catalogue #120-17) fortwo days followed by treatment with DMEM/F12 media supplemented with 2%FBS, 100 ng/ml activin A (AA) and +/−10 or 20 ng/ml of WNT-1 for anadditional three days. The following combinations of WNT1+AA weretested:

a) 20 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2followed by 2% FBS+DM-F12+100 ng/ml AA for day three, b) 20 ng/ml ofWNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2%FBS+DM-F12+100 ng/ml AA for days 3-5, c) 10 ng/ml of WNT1+100 ng/ml AAin 0.5% FBS+DM-F12 for days 1-2 followed by 2% FBS+DM-F12+100 ng/ml AAfor day three, d) 10 ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 fordays 1-2 followed by 2% FBS+DM-F12+100 ng/ml AA for days 3-5, e) 20ng/ml of WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by2% FBS+DM-F12+100 ng/ml AA+20 ng/ml of WNT1 for day three, f) 20 ng/mlof WNT1+100 ng/ml AA in 0.5% FBS+DM-F12 for days 1-2 followed by 2%FBS+DM-F12+100 ng/ml AA+20 ng/ml of WNT1 for days 3-5. FIGS. 49A and 49Bdisplays the real-time PCR data for definitive endoderm markersfollowing treatment of the H1 cells with low serum, AA and Wnt-1.Addition of 20 ng/ml of Wnt1 in the present of 100 ng/ml of AA resultedin significant upregulation of definitive endoderm markers (Bry, CXCR4,GSC, SOX17, HNF-3B, and GATA-4).

Publications cited throughout this document are hereby incorporated byreference in their entirety. Although the various aspects of theinvention have been illustrated above by reference to examples andpreferred embodiments, it will be appreciated that the scope of theinvention is defined not by the foregoing description but by thefollowing claims properly construed under principles of patent law.

TABLE IA LIST OF PRIMARY ANTIBODIES USED FOR FACS ANDIMMUNOSTAINININGANALYSIS. Antibody Supplier Isotype Clone SSEA-1Chemicon (CA) Mouse IgM MC-480 SSEA-3 Chemicon (CA) Mouse IgG3 MC-631SSEA-4 Chemicon (CA) Rat IgM MC-813-70 TRA 1-60 Chemicon (CA) Mouse IgMTRA 1-60 TRA 1-81 Chemicon (CA) Mouse IgM TRA 1-81 TRA 1-85 Chemicon(CA) Mouse IgG1 TRA 1-85 AP R&D Systems Mouse IgG1 B4-78 HNF3β R&DSystems Goat IgG PDX1 Santa Cruz Goat IgG A-17 Biotechnology, INC GATA4R&D Systems Goat IgG Sox 17 R&D Systems Goat IgG CD 9 BD Mouse IgG1M-L13

TABLE IB LIST OF SECONDARY CONJUGATED ANTIBODIES USED FOR FACS ANDIMMUNOSTAINININGANALYSIS. Secondary Conjugated Antibody SupplierDilution Goat Anti-Mouse IgG Jackson 1:200 APC conjugated ImmunoResearch(PA) Goat Anti-Mouse IgG Jackson 1:200 PE conjugated ImmunoResearch (PA)Donkey anti-rabbit PE Jackson 1:200 or -APC conjugated ImmunoResearch(PA) Donkey anti-goat PE or Jackson 1:200 -APC conjugated ImmunoResearch(PA) Goat anti-mouse IgM SouthernBiotech (AL) 1:200 PE Goat anti-Rat IgMPE SouthernBiotech (AL) 1:200 Goat anti-mouse IgG3 SouthernBiotech (AL)1:200 PE

TABLE IIA CHANGES IN PROTEIN EXPRESSION IN HUMAN EMBRYONIC STEM CELLSWITH TIME, FOLLOWING ACTIVIN A TREATMENT. 0-DAY 2-DAY 5-DAY 8-DAY SSEA-398.67% 92.14%  42.9% 22.05% CD9 92.64% 29.42%  7.27%  4.1% ECAM 61.23%20.87% 14.17%  1.02% NCAM  7.33%  5.04%  21.1%  8.86% CXCR4  8.53% 20.2% 55.26% 56.92%

TABLE IIB CHANGES IN PROTEIN EXPRESSION IN HUMAN EMBRYONIC STEM CELLSWITH TIME, FOLLOWING ACTIVIN A TREATMENT. 1-day 3-day 5-day AA AA AAUntreated 100 ng/ml Untreated 100 ng/ml Untreated 100 ng/ml CXCR4+   13%    6%  7.6%    38% 3%  65.5% CXCR4 + C-Kit+ 5.32%  2.97%  2.9% 31.56%3% 55.21% CXCR4 + EPCAM+ 11.5% 14.58% 5.26% 36.67% 3%  54.5% CXCR4 +CD9+ 12.27%   8.13% 2.72% 24.11% 3%  2.1%

TABLE IIC CHANGES IN PROTEIN EXPRESSION IN HUMAN EMBRYONIC STEM CELLSWITH TIME, FOLLOWING ACTIVIN A TREATMENT. 5-day AA treatment CXCR4+92.78% CXCR4+/C-kit+ 92.90% CXCR4+/EPCAM 87.99% CXCR4+/CD99+ 88.78%CXCR4+/CD9+  7.03%

TABLE III EXPRESSION PROFILE OF PLURIPOTENCY MARKERS FOR THE EMBRYONICSTEM CELL USED IN THE PRESENT INVENTION. H9 Marker FACS RT-PCR StainingOCT3/4 + + SOX-2 + UTF-1 + REX-1 + hTERT + Cx 43 + Cx 45 + ABCG-2 +SSEA-1 ± (36.35%) SSEA-3 + (94.38%) + SSEA-4 + (98.77%) + TRA-1-81 +(85.85%) + TRA-1-60 + (78.14%) + TRA-1-85 + (95.96%) CD9 + (92.02%) AP +(99%) +

TABLE IV DIFFERENTIAL EXPRESSION OF GENES BETWEEN UNDIFFERENTIATEDEMBRYONIC STEM CELLS AND DEFINITIVE ENDODERM STAGE CELLS CULTURED ONEITHER MATRIGELTM OR MOUSE EMBRYONIC FIBROBLASTS AFTER 5 DAYS OFTREATMENT. H9P83 on MEFs-DE Gene H9P83 on Matrigel- DE H9P44 on H9P44 onIdentifier Gene Title Gene ID M trigel SEM stage SEM MEFs SEM stage SEMD87811 Homo GATA6 −2.12 0.19 2.82 2.20 −2.56 0.46 5.34 1.79 sapiens mRNAfor GATA-6, complete cds./ PROD = GATA-6/ FL = gb: U66075.1 gb:NM_005257.1 gb: D87811.1 AW157548 insulin-like IGFBP5 −3.28 0.17 3.312.11 −3.78 0.36 5.35 2.00 growth f ctor binding protein 5/ FL = gb:M65062.1 gb: M62782.1 gb: NM_000599.1 gb: AF055033.1 NM_001898 Homo CST4−2.15 1.26 2.54 1.95 −2.71 0.98 4.64 1.63 sapiens cystatin SN (CST1),mRNA./ PROD = cysttin SN/FL = gb: J03870.1 gb: NM_001898.1 AK000680 HomoPAG1 −2.87 0.91 1.61 0.22 −4.08 0.50 1.68 0.10 apien cDNA FLJ20673 fis,clone KAIA4464./ FL = gb: AF240634.1 gb: NM_018440.1 NM_022642 Homo —−2.24 0.12 2.97 0.42 −3.78 0.07 2.51 0.44 sapiens chorionicsomatomammotropin hormone 1 (placental lactogen) (CSH1), transcriptvariant 4, mRNA./PROD = chorionic somatomammotropin hormone 1, isoform4/FL = gb: NM_022642.1 NM_001317 Homo CSH1 −2.95 0.57 2.69 0.36 −4.04 0.511.86 0.68 sapiens chorionic somatomam motropin hormone 1 (placentallactogen) (CSH1), transcript variant 1, mRNA./ PROD = chorionicsomatomam- motropin hormone 1, isoform 1precursor FL = gb: NM_001317.2gb: J00118.1 BC005921 Homo sapiens, CSH1 −2.26 0.09 3.26 0.23 −2.96 0.372.58 0.45 chorionic somatomammotropin hormone 1 (placental lactogen),clone MGC: 14518, mRNA, complete cds./ PROD = chorionicsomatomammotropin hormone 1 (placentallactogen)/ FL = gb: BC005921.1AI796169 GATA-binding GATA3 −4.45 0.10 0.24 1.30 −4.72 0.13 0.80 2.05protein 3/ FL = gb: NM_002051.1 gb: M69106.1 gb: BC003070.1 NM_020991Homo sapiens CSH1 −1.27 0.48 3.19 0.23 −2.91 0.35 2.62 0.54 chorionicsomatomammotropin hormone 2 (CSH2), transcript variant 1, mRNA./ PROD =chorionic somatomammotropin hormone 2, isoform 1precursor/ FL = gb:NM_020991.2 gb: BC002717.1 NM_021827 Homo sapiens CCDC81 −0.37 0.35 3.162.05 −2.02 1.27 5.25 1.98 hypothetical protein FLJ23514 (FLJ23514),mRNA./ PROD = hypothetical protein FLJ23514/ FL = gb: NM_021827.1AB028021 Cluster Incl. FOXA2 −2.97 0.25 0.59 3.25 −3.43 0.57 4.12 2.57AB028021: Homo sapiens HNF-3beta mRNA for hepatocyte nuclear factor-3beta, complete cds/cds = (196,1569)/ gb = AB028021/ gi = 4958949/ ug =Hs. 155651/ len = 1944 NM_002521 Homo NPPB 1.54 0.11 5.47 1.17 −0.150.38 6.24 1.23 sapiens natriuretic peptide precursor B (NPPB), mRNA./PROD = natriuretic peptide precursor B/ FL = gb: NM_002521.1 b: M25296.1AA352113 ESTs ST8SIA4 −4.01 1.24 −0.99 2.04 −4.79 1.00 1.05 1.62BM128432 Homo IGFBP5 −2.73 1.11 2.31 2.30 −3.48 0.56 4.45 2.02 sapiensfull length insert cDNA clone YA81B05 NM_002770 Homo PRSS1 −2.77 0.331.59 2.68 −3.13 0.48 3.88 2.95 sapiens protease, serine, 2 (trypsin 2)(PRSS2), mRNA./ PROD = protease, serine, 2 (trypsin 2)/ FL = gb:M27602.1 gb: NM_002770.1 NM_022579 Homo CSH1 −1.58 0.91 2.48 0.38 −3.330.13 1.77 0.49 sapiens chorionic somatomammotropin hormone-like 1(CSHL1), transcript variant 3, mRNA./ PROD = chorionic somatomammotropinhormone-like 1, isoform 3 precursor/ FL = gb: NM_022579.1 NM_005454 HomoCER1 2.82 0.09 5.78 1.04 1.48 0.05 6.74 1.18 sapiens cerberus 1 (Xenopuslaevis) homolog (cysteine knot uperfamily) (CER1), mRNA./ PROD =cerberus 1/ FL = gb: NM_005454.1 NM_022645 Homo CSH1 −2.30 0.33 2.950.31 −2.78 0.24 2.45 0.34 sapiens chorionic somatomammotropin hormone 2(CSH2), transcript variant 3, mRNA./ PROD = chorionic somatomammotropinhormone 2, isoform 3precursor/ FL = gb: NM022645.1 AI821586 ESTs,LOC440981 −3.22 0.97 0.66 3.19 −2.97 0.13 4.22 2.76 Moderately similarto JE0284 Mm- 1 cell derived transplantability- associated protein 1b(H.sapiens) AL121722 Human DNA — −2.95 0.36 −0.01 2.69 −3.43 0.38 2.952.66 sequence from clone RP4-788L20 on chromosome 20 Contains the HNF3B(hepatocyte nuclear factor 3, beta) gene. a novel gene based on ESTs,ESTs, and CpG STSs, GSSs and CpG Islands NM_001311 Homo CRIP1 1.66 0.171.90 0.07 −2.96 0.66 1.80 0.24 sapiens cysteine-rich protein 1(intestinal) (CRIP1), mRNA./ PROD = cysteine-rich protein 1(intestinal)/ FL = gb: U58630.1 gb: BC002738.1 gb: NM_001311.1 gb:U09770.1 AY177407 Homo GSC −4.59 0.18 −1.08 2.89 −4.64 0.06 1.89 2.79sapiens homeobox protein goosecoid mRNA, complete cds./PROD = homeoboxprotein goosecoid/ FL = gb: AY177407.1 gb: NM_173849.1 NM_005442 HomoEOMES −0.16 0.29 2.89 1.70 0.13 0.16 4.90 1.34 sapiens eomesoderm in(Xenopus laevis) homolog (EOMES) mRNA. PROD = eomesodermin (Xenopuslaevis) homolog/ FL = gb: AB031038.1 gb: NM_005442.1 L01639 Human CXCR40.64 0.26 3.71 1.78 −0.16 0.50 5.48 1.77 (clone HSY3RR) neuropeptide Yreceptor (NPYR) mRNA, complete cds./PROD = neuropeptide Y receptor/ FL =gb: L06797.1 gb: NM_003467.1 gb: AF025375.1 gb: AF147204.1 gb: M99293.1gb: L01639.1 NM_022646 Homo — −1.57 0.60 2.67 0.26 −1.88 0.98 2.22 0.35sapiens chorionic somatomammotropin hormone 2 (CSH2), transcript variant4, mRNA./ PROD = chorionic somatomammotropin hormone 2, isoform4/FL =gb: NM_22646.1 AW 7532 Human IGFBP5 .31 .25 4.59 1.53 .72 .09 6.19 1.58insulin-like growth factor binding protein 5 (IGFBP5) mRNA NM_002160Homo TNC −0.24 0.29 2.23 0.80 −0.81 .81 2.85 0.82 sapiens hexabrachion(tenascin C, cytotactin) (HXB), mRNA./ PROD = hexabrachion (tenascin C,cytotactin)/ FL = gb: M55618.1 gb: NM_002160.1 AA149250 ESTs, LOC6456381.27 0.61 4.23 1.26 −0.64 .40 2.47 1.23 Weakly similar to WDNM RAT WDNM1PROTEIN PRECURSOR (R.norvegicus) AW977527 ESTs — −0.91 0.99 1.18 0.68−2.52 1.01 1.59 0.64 NM_022454 Homo SOX17 −1.01 0.33 2.29 2.08 −0.14 .154.60 1.73 sapiens hypothetical protein FLJ22252 similar to SRY-boxcontaining gene 17 (FLJ22252), mRNA./ PROD = hypothetical proteinLJ22252 similar to SRY- boxcontaining gene 17/FL = gb: NM_022454.1AI640307 protocadherin 10 PCDH10 −1.89 1.37 1.53 1.32 −1.33 0.38 2.991.19 AJ224869 Homo — 0.98 0.18 4.22 1.88 1.34 0.17 6.36 1.49 sapiensCXCR4 gene encoding receptor CXCR4 AI824037 ESTs, FREM1 −1.42 0.36 1.221.95 −1.37 0.61 3.48 1.49 Weakly similar to FCE2 MOUSE LOW AFFINITYIMMUNO- GLOBULIN EPSILON FC RECEPTOR (M.musculus) BE222344 splicing —0.50 0.05 3.01 0.93 −0.94 1.18 4.27 0.94 factor, arginineserine-rich 5NM_001643 Homo APOA2 −2.25 1.05 1.72 2.60 −1.20 0.44 4.47 2.42 sapiensapolipoprotein A-II (APOA2), mRNA./ PROD = apolipopprotein A-IIprecursor/FL = gb: M29882.1 gb: NM_001643.1 gb: BC005282.1 I821669 ESTs— −0.94 0.79 1.71 2.19 −0.89 0.21 3.91 1.94 NM_002608 Homo PDGFB −0.230.82 1.87 0.15 −2.27 0.65 1.92 0.13 sapiens platelet-derived growthfactor beta polypeptide (simian sarcoma viral (v-sis) oncogene homolog)(PDGFB), mRNA./PROD = platelet-derived growth factor beta poly-peptide(simian sarcoma viral (v-sis) oncogene homolog)/ FL = gb:M12783.1 gb: NM_002 AW444761 ESTs CDKN2B −3.35 0.90 0.39 0.52 −3.11 0.880.80 0.25 BF223214 ESTs — −0.48 0.08 1.69 2.04 −1.51 0.25 4.09 1.98AF154054 Homo GREM1 1.68 0.26 4.61 0.74 0.62 0.04 3.58 0.87 sapiens DRM(DRM) mRNA, complete cds./ PROD = DRM/ FL = gb: NM_013372.1 gb:AF110137.2 gb: AF045800.1 gb: AF154054.1 NM_021223 Homo MYL7 1.81 0.054.28 0.81 0.17 0.08 4.87 0.96 sapiens yosin light chain 2a (LOC58498),mRNA./ PROD = myosin light chain 2a/ FL = gb: NM_021223.1 AI817041 Gprotein- CMKOR1 −0.19 0.27 2.67 1.97 0.06 0.18 5.05 1.64 coupledreceptor NM_003670 Homo sapiens BHLHB2 1.09 0.08 3.85 0.10 −0.11 0.173.46 0.03 basic helix- loop-helix domain containing, class B, 2(BHLHB2), mRNA./PROD = differentiated embryo chondrocyte expressedgene1/FL = gb: AB004066.1 gb: NM_003670.1 NM_023915 Homo sapiens G GPR87−1.70 0.61 1.64 0.18 −2.99 0.06 1.40 0.23 protein-coupled receptor 87(GPR87), mRNA./PROD = G proteincoupled receptor 87/ FL = gb: NM_023915.1gb: AF237763.1 NM_003867 Homo sapiens FGF17 −3.05 0.39 0.03 2.07 −2.130.41 2.49 1.35 fibroblast growth factor 17 (FGF17), mRNA./PROD =fibroblast growth factor 17/ FL = gb: NM_003867.1 gb: AB009249.1NM_024426 Homo WT1 −3.23 0.37 −1.11 0.62 −4.20 0.56 −2.44 1.26 sapiensWilms tumor 1 (WT1), transcript variant D, mRNA./ PROD = Wilms tumor 1isoform D/ FL = gb: NM_024424.1 gb: NM_024426.1 NM_033136 Homo FGF1−3.10 1.42 0.09 0.83 −3.16 0.63 −0.78 1.21 sapiens fibroblast growthfactor 1 (acidic) (FGF1), transcript variant 2, mRNA./ PROD = fibroblastgrowth factor 1 (acidic) isoform 2precursor/ FL = gb: NM_033137.1 gb:NM_033136.1 X99268 H.sapiens TWIST1 0.10 0.33 3.94 0.24 0.34 0.22 3.450.34 mRNA for B- HLH DNA binding rotein./ PROD = B-HLH DNA bindingprotein/ FL = gb: NM_000474.1 AL524520 G protein- LGR5 −2.27 1.43 0.761.40 −1.58 0.40 2.51 1.35 coupled receptor 49 NM_022557 Homo CSH1 −0.910.19 1.40 0.22 −2.12 0.08 1.47 0.14 sapiens growth hormone 2 (GH2),transcript variant 2, mRNA./ PROD = growth hormone 2, isoform 2precursor/ FL = gb: J03756.1 gb: NM_022557.1 AL544576 ESTs TMEM88 −1.960.68 1.75 0.58 −1.45 0.86 2.08 0.70 NM_022580 Homo sapiens CSH1 −1.200.86 2.30 0.39 −1.78 0.64 1.55 0.63 chorionic somatomammotropinhormone-like 1 (CSHL1), transcript variant 4, mRNA./ PROD = chorionicsomatomammotropin hormone-like 1, isoform 4/ FL = gb: NM_022580.1 J03580Human, PTHLH −2.72 0.33 −0.80 0.40 −4.05 0.41 −1.42 0.73 parathyroid-like protein associated with humoral hypercalcemia of malignancy) mRNA,complete cds./ FL = gb: J03580.1 BC029835 Homo LOC646867 −2.66 1.12 1.011.85 −1.40 0.41 3.03 1.61 sapiens, clone IMAGE: 5169 759, mRNA AI452798ESTs MYOCD 0.98 0.13 3.31 0.66 −0.07 0.28 2.80 0.98 NM_022559 Homosapiens CSH1 −1.56 0.38 2.00 0.32 −2.07 0.42 1.47 0.28 growth hormone 1(GH1), transcript variant 2, mRNA./ PROD = growth hormone 1, isoform 2precursor/ FL = gb: NM_022559.1 NM_001318 Homo sapiens CSH1 0.15 0.412.83 0.40 −1.30 0.34 2.37 0.50 chorionic somatomammotropin hormone-like1 (CSHL1), transcript variant 1, mRNA./ PROD = chorionicsomatomammotropin hormone-like 1, isoform 1/ FL = gb: NM_001318.2 M65062Human IGFBP5 −2.80 1.17 2.19 2.00 −0.99 0.23 4.11 1.81 insulin-likegrowth factor binding protein 5 (IGFBP-5) mRNA, complete cds./ PROD =insulin-like growth factor binding protein 5/ FL = gb: M650 gb:M6262.1782.1 gb: NM_000599.1 gb: AF055033.1 AF207990 Homo FER1L3 0.490.40 3.00 0.81 −0.16 0.09 4.02 0.92 sapiens fer-1 like protein 3(FER1L3) mRNA, complete cds./ PROD = fer-1 like protein 3/ FL = gb:AF207990.1 AI079944 ESTs — −0.78 0.22 0.08 0.59 −3.56 0.24 −0.13 0.73BC003070 Homo GATA3 1.07 0.04 3.23 0.96 0.31 0.26 4.45 0.80 sapiens,GATA- binding protein 3, clone MGC: 2346, mRNA, complete cds./ PROD =GAT A-binding protein 3/ FL = gb: NM_002051.1 b: M69106.1 gb: BC003070.1BE877796 collagen, COL8A1 −3.76 1.17 −1.28 0.73 −4.89 0.54 −1.33 1.64type VIII, alpha 1/ FL = gb: NM_001850.1 NM_022560 Homo CSH1 −2.23 0.741.95 0.08 −2.01 0.51 1.47 0.31 sapiens growth hormone 1 (GH1),transcript variant 3, mRNA./ PROD = growth hormone 1, isoform 3precursor/ FL = gb: NM_022560.1 BE328496 hypothetical — −0.59 0.25 1.760.13 −2.01 0.97 1.73 0.26 protein PRO2032/ FL = gb: AF116683.1 gb:NM_018615.1 NM_022469 Homo GREM2 −1.15 0.25 0.09 0.88 −3.43 0.90 0.760.47 sapiens hypothetical protein FLJ21195 similar to protein related toDAC and cerberus (FLJ21195), mRNA./ PROD = hypothetical protein FLJ21195similar to proteinrelated/ to DAC nd cerberus FL = gb: NM022469.1NM_001362 Homo DIO3 −1.73 0.70 1.99 1.92 −1.05 0.79 4.23 1.51 sapiensdeiodinase, iodothyronine, type III (DIO3), mRNA./PROD = thyroxinedeiodinase type III/FL = gb: NM_001362.1 gb: S79854.1 NM_022581 HomoCSH1 −1.56 0.67 2.08 0.42 −1.63 0.80 1.47 0.53 sapiens chorionicsomatomammotropin hormone-like 1 (CSHL1), transcript variant 5,mRNA./PROD = chorionic somatomammotropin hormone-like 1, isoform 5precursor/FL= gb: NM_022581.1 NM_013372 Homo GREM1 1.61 0.07 4.15 0.640.91 0.15 3.20 0.81 sapiens cysteine knot superfamily 1, BMP antagonist1 (CKTSF1B1), mRNA./PROD = cysteine knot superfamily 1, BMP ntagonist 1/FL = gb: 013372.1 gb: AF110137.2 gb: AF045800.1 gb: AF154054.1 NM_022561Homo CSH1 −1.32 0.60 1.91 0.25 −1.66 0.46 1.57 0.22 sapiens growthhormone 1 (GH1), transcript variant 4, mRNA./ PROD = growth hormone 1,isoform 4 precursor/ FL = gb: NM_022561.1 NM_022659 Homo — −1.58 0.75−1.24 1.79 −3.62 0.20 −1.81 1.25 sapiens hypothetical protein FLJ11500similar to EBF2 (FLJ11500), mRNA./PROD = hypothetical protein FLJ11500similar to EBF2/FL = gb: NM_022659.1 AF006060 Homo CSH1 −1.21 0.06 1.130.05 −3.08 0.63 0.50 0.17 sapiens placental growth hormone 20kDa isoform(hGH-V) RNA, complete cds./ PROD = placental growth hormone 20kDaisoform/ FL = gb: AF006060.1 gb: NM_022556.1 AI688418 plexin A2 PLXNA2−0.18 0.14 0.84 1.58 −1.28 0.53 2.74 0.96 M86849 Homo — −5.08 0.26 0.540.47 −1.64 0.19 −0.09 0.44 sapiens connexin 26 (GJB2) mRNA, completecds./PROD = connexin 26/ FL = gb: NM_004004.1 gb: M86849.2 N71923fibronectin — 0.30 0.24 2.83 1.46 0.53 0.12 4.38 1.46 leucine richtransmembrane protein 3/ FL = gb: AF169677.1 gb: NM_013281.1 NM_013281Homo FLRT3 −0.20 0.33 2.14 1.61 0.00 0.15 3.96 1.39 sapiens fibronectinleucine rich transmembrane protein 3 (FLRT3), mRNA./ PROD = fibronectinleucine rich transmembrane protein3/ FL = gb: AF169677.1 b: NM_013281.1AI601101 Homo sapiens FAM84A 0.46 0.38 3.07 0.46 −0.32 0.46 3.82 0.48CDNA: FLJ21410 fis, clone COL03938 NM_000325 Homo PITX2 1.37 0.17 3.510.47 0.89 0.16 4.02 0.47 sapiens paired-like homeodomain transcriptionfactor 2 (PITX2), mRNA./ PROD = paired-like homeodomain transcriptionfactor 2/ FL = gb: NM_000325.1 gb: U69961.1 gb: AF048720.1 AI692659 heatshock PRDM1 0.21 0.25 1.67 0.77 −0.67 0.17 2.61 0.80 90kD protein 1,alpha NM_000602 Homo SERPINE1 2.97 0.16 4.75 1.89 1.55 0.22 2.75 1.87sapiens serine (or cysteine) proteinase inhibitor, cladeE (nexin,plasminogen activator inhibitor type 1), member 1 (SERPINE1), mRNA./PROD= serine or ysteine) proteinase inhibitor cladeE (nexin, plasminogenactivator inhibitor type 1), membe NM_001480 Homo GALR1 −1.90 0.68 0.040.73 −2.42 0.72 −0.34 0.73 sapiens galanin receptor 1 (GALR1), mRNA./PROD = galanin receptor 1/ FL = gb: NM_001480.2 gb: U23854.1 gb:L34339.1 gb: U53511.1 NM_000393 Homo COL5A2 4.29 0.14 5.25 0.21 2.620.02 5.48 0.13 sapiens collagen, type V, alpha 2 (COL5A2), mRNA./PROD =collagen, type V, alpha 2/FL = gb: NM_000393.1 N63706 ESTs I 0.06 0.181.73 1.89 −0.74 0.13 3.75 1.75 AF132818 Homo KLF5 0.63 0.11 3.16 0.500.07 0.42 3.40 0.44 sapiens colon Kruppel-like factor (CKLF) mRNA,complete cds./PROD = colon Kruppel- like factor/ FL = gb: AF13818.1 gb:AF287 72.1 gb: AB030824.1 gb: NM_001730.1 gb: D14520.1 X59065 H.sapiens— −1.17 1.11 −0.39 1.06 −2.61 0.26 −0.96 1.01 FGF gene, exon 3/FL = gb:NM_000800.1 gb: M13361.1 R73554 Human IGFBP5 −0.12 0.15 2.63 1.41 −0.690.16 4.00 1.65 insulin-like growth factor binding protein 5 (IGFBP5)mRNA NM_002149 Homo HPCAL1 −0.18 0.28 1.81 0.44 −1.98 0.86 1.18 0.22sapiens hippocalcin- like 1 (HPCAL1), mRNA./PROD = hippocalcin-like1/ FL= gb: NM_002149.1 gb: D16227.1 AI093327 ESTs — 0.69 0.11 3.09 0.58 0.450.07 2.29 0.85 NM_003240 Homo PYCR2 6.22 0.16 6.97 0.60 4.16 0.07 7.510.68 sapiens endometrial bleeding associated factor (left- rightdetermination, factor A; transforming growth factor beta superfamily)(EBAF), mRNA. PROD = transforming growth factor, beta 4/ FL = gb:U81523.1 gb: NM_003240.1 gb: AF081513.1 AI263909 ras homolog RHOB 3.890.16 5.88 0.13 3.01 0.15 5.64 0.10 gene family, member B/ FL = gb:NM_004040.1 NM_001792 Homo CDH2 3.83 0.17 6.01 0.11 2.85 0.13 5.74 0.17sapiens cadherin 2, type 1, N- cadherin (neuronal) (CDH2), mRNA./ PROD =cadherin 2, type 1, N-cadherin (neuronal)/ FL = gb: NM_001792.1 gb:M34064.1 NM_003897 Homo IER3 5.45 0.15 6.97 0.17 4.33 0.11 6.75 0.27sapiens immediate early response 3 (IER3), mRNA./ PROD = immediate earlyresponse 3/ FL = gb: BC005080.1 gb: BC000844.1 gb: AF083421.1 gb:NM_003897.1 AF278532 Homo NTN4 −0.16 0.30 1.70 0.91 −0.67 0.48 2.66 0.74sapiens beta-netrin mRNA, complete cds./ PROD = beta-netrin/ FL = gb:AF119916.1 gb: AF297711.1 gb: NM_021229.1 gb: AF278532.1 AF348491 HomoCXCR4 1.39 0.21 4.05 1.58 1.45 0.07 5.67 1.65 sapiens chemokine receptorCXCR4 mRNA, complete cds./PROD = chemokine receptor CXCR4/ FL = gb:AF348491.1 NM_030781 Homo COLEC12 1.93 0.13 3.64 1.66 1.96 0.18 5.681.51 sapiens scavenger receptor with C-type lectin (SRCL), mRNA./ PROD =scavenger receptor with C-type lectin/ FL = gb: NM_030781.1 NM_000599Homo IGFBP5 −0.31 0.34 3.03 1.85 0.12 0.24 4.51 1.75 sapiensinsulin-like growth factor inding protein 5 (IGFBP5), mRNA./ PROD =insulin- like growth factor binding protein 5/ FL = gb: M65062.1 gb:M62782.1 gb: NM_000599.1 gb: AF055033.1 AI348094 KIAA0882 TBC1D9 0.130.26 3.21 1.13 0.96 0.13 4.67 1.08 protein BG287862 AHNAK AHNAK 1.470.20 3.72 0.51 1.37 0.17 4.34 0.58 nucleoprotein (desmoyokin) AI676059ESTs FOXQ1 −0.32 0.55 3.08 1.63 0.50 0.17 4.81 1.47 AI127440 ESTs —−0.85 0.36 0.60 1.22 −0.65 0.27 2.13 1.13 AL574210 serine (or SERPINE12.81 0.24 5.07 0.88 2.13 0.15 3.96 0.86 cysteine) proteinase inhibitor,clade E (nexin, plasminogen activator inhibitor type 1), member 1/ FL =gb: NM000602.1 gb: M16006.1 AB037810 Homo SIPA1L2 3.88 0.04 5.66 0.083.18 0.11 5.85 0.11 sapiens mRNA for KIAA1389 protein, partial cds./PROD = KIAA1389 protein M_001394 Homo DUSP4 0.22 0.37 2.88 1.09 0.500.14 4.12 0.99 sapiens dual specificity phosphatase 4 (DUSP4), mRNA./PROD = dual specificity phosphatase 4/ FL = gb: NM_001394.2 gb:BC002671.1 gb: U48807.1 gb: U21108.1 BC029442 Homo — 2.04 0.20 3.80 0.741.18 0.05 4.50 0.65 sapiens, Similar to immunity associated protein 1,clone MGC: 32707 IMAGE: 4618467, mRNA, complete cds./PROD = Similar toimmunity associated protein 1/ FL = gb: BC029442.1 NM_000700 Homo ANXA15.00 0.18 6.27 1.28 3.67 0.05 4.96 1.25 sapiens annexin A1 (ANXA1),mRNA./ PROD = annexin I/ FL = gb: BC001275.1 gb: NM_000700.1 BC000740Homo CCKBR 1.08 0.36 3.93 1.84 1.93 0.03 5.86 1.82 sapiens,holecystokinin B receptor, clone MGC: 2199, mRNA, complete cds./PROD =cholecystokinin B receptor/ FL = gb: L07746.1 gb: L08112.1 gb: S70057.1gb: BC000740.1 gb: L04473.1 gb: NM_000731.1 N36408 hypothetical FOSL2−0.09 0.40 2.07 0.04 −0.85 0.24 1.71 0.28 protein FLJ23306/ FL = gb:NM_024530.1 AF072242 Homo MBD2 −3.98 0.38 −0.59 0.27 −3.07 0.22 −1.700.68 sapiens methyl-CpG binding protein MBD2 (MBD2) mRNA, completecds./PROD = methyl-CpG binding protein MBD2/ FL = gb: NM_003927.2 gb:AF072242.1 AF211891 Homo MIXL 1 −0.63 0.38 0.77 0.95 −2.29 0.44 0.921.71 sapiens Mix- like homeobox rotein 1 (MILD1) mRNA, completecds./PROD = Mix-like homeobox protein 1/ FL = gb: AF211891.1 BF063186ESTs CALD1 1.16 0.19 2.03 0.42 −1.20 1.02 1.65 0.27 NM_000362 Homo TIMP30.08 0.32 1.98 0.09 −0.34 0.33 1.80 0.26 sapiens tissue inhibitor ofmetalloproteinase 3 (Sorsby fundus dystrophy, pseudoinflammatory)(TIMP3), mRNA./PROD = tissue inhibitor of metalloproteinase 3precursor/FL = gb: NM_000362.2 gb: U14394.1 gb: U67195.1 gb: U02571.1 AK022852Homo SIPA1L2 2.87 0.12 4.50 0.09 1.87 0.01 4.55 0.09 sapiens CDNAFLJ12790 fis, clone NT2RP2001 985, weakly similar to Homo sapienshigh-risk human papilloma iruses E6 oncoproteins targeted protein E6TP1alpha mRNA. BE500942 Homo C6orf155 3.23 0.10 4.68 0.86 1.95 0.03 3.770.96 sapiens mRNA; cDNA DKFZp761M0111 (from clone DKFZp761M0111)AW665892 paternally MFAP5 −1.82 0.54 0.37 0.60 −1.54 0.39 −0.01 0.28expressed 3 AK025063 Homo FAM84A −1.34 0.84 0.75 0.45 −2.35 0.85 1.010.53 sapiens cDNA: FLJ21410 fis, clone COL03938. NM_001828 Homo CLC 2.170.21 3.53 1.07 0.45 0.08 2.11 1.27 sapiens Charot- Leyden crystalprotein (CLC), mRNA./ PROD = Charot- Leyden crystal protein/FL = gb:NM_001828.3 gb: L01664.1 M15329 Human IL1A 0.85 0.32 2.72 0.30 −0.880.60 2.20 0.22 interleukin 1- alpha (IL1A) mRNA, complete cds./PROD =interleukin 11 alpha/FL = gb: M1531 BC002671 Homo DUSP4 1.7 0.03 4.301.3 2.10 0.10 5.60 1.22 sapiens, dual specificity phosphatase 4, cloneMGC: 3713, mRNA, complete cds./PROD = dual specificity phosphatase 4/ FL= gb: NM_001394.2 gb: BC002671.1 gb: U48807.1 gb: U21108.1 AA524250deleted in DLC1 1.02 0.05 2.35 0.98 0.10 0.38 3.26 0.96 liver cancer 1BC001211 Homo KIFC3 −1.53 0.72 0.81 0.38 −2.08 0.42 0.75 0.16 sapiens,kinesin family member C3, clone MGC: 3226, mRNA, complete cds./PROD =kinesin family member C3/ FL = gb: BC001211.1 gb: NM_005550.1 gb:AF004426.1 NM_004560 Homo sapiens ROR2 0.76 0.08 2.20 0.81 0.08 0.143.06 0.95 receptor tyrosine kinase-like orphan receptor 2 (ROR2),mRNA./PROD = receptor tyrosine kinase-like orphan receptor 2/FL = gb:M97639.1 gb: NM_004560.1 BC000125 Homo TGFB1 1.16 0.18 3.43 0.08 0.720.14 3.30 0.16 sapiens, Similar to transforming growth factor, beta 1,clone MGC: 3119, mRNA, complete cds./PROD = Similar to transforminggrowth factor, beta 1/ FL = gb: M38449.1 gb: BC001180.1 gb: BC000125.1gb: NM_000660.1 NM_016931 Homo NOX4 1.83 0.06 3.31 1.29 1.20 0.14 2.281.27 sapiens NADPH oxidase 4 (NOX4), mRNA./PROD = NADPH oxidase 4/ FL =gb: AF261943.1 gb: NM_016931.1 gb: AF254621.1 gb: AB041035.1 BC001830Homo TGFB I1 0.95 0.17 3.59 0.73 .52 0.09 2.74 0.69 sapiens, Similar totransforming growth factor beta 1 induced transcript 1, clone MGC: 4078,mRNA, complete cds./PROD = Similar to transforming growth factor beta1induced transcript 1/FL = gb: NM_015927.1 gb: BC001830.1 gb: AF116343.1NM_024576 Homo OGFRL1 3.15 0.13 4.49 0.82 .63 0.06 3.30 0.98 sapienshypothetical protein FLJ21079 (FLJ21079), mRNA./PROD = hypotheticalprotein FLJ21079/ FL = gb: NM_024576.1 NM_001963 Homo EGF 0.12 0.22 1.821.15 −0.62 0.36 2.68 1.23 sapiens epidermal growth factorbeta-urogastrone) (EGF), mRNA./ PROD = epidermal growth factor (beta-urogastrone)/ FL = gb: NM_001963.2 BE620374 ESTs C6orf155 1.97 0.05 3.351.01 0.59 0.17 2.10 1.05 AL359062 Homo COL8A1 −1.31 0.25 2.32 0.94 0.030.12 3.51 1.02 sapiens mRNA full length insert cDNA clone EUROIMAGE1913076. AL117653 Homo MITF −0.12 0.61 2.32 0.10 0.21 0.13 2.56 0.06sapiens mRNA; CDNA DKFZp586C 0224 (from clone DKFZp586C 0224). AL021977Cluster Incl. — 1.92 0.14 4.53 0.43 2.15 0.12 4.66 0.22 AL021977:bK447C4.1 (novel MAFF (v-maf musculoaponeurotic fibrosarcoma (avian)oncogene family, protein F) LIKE protein)/ cds = (0.494)/ gb = AL021977/gi = 4914526/ ug = Hs.51305/ len = 2128 NM_003564 Homo TAGLN2 5.43 0.126.76 0.06 3.48 0.27 6.69 0.04 sapiens transgelin 2 (TAGLN2), mRNA./PROD= transgelin 2/ FL = gb: D21261.1 gb: NM_003564.1 BC005107 Homo — 7.080.07 6.42 0.26 2.60 0.07 6.93 0.22 sapiens, clone IMAGE: 3840937, mRNA,partial cds./ PROD = Unknown (protein for IMAGE: 3840937) NM_001124 HomoADM 4.70 0.22 7.61 0.19 5.21 0.06 7.58 0.21 sapiens adrenomedullin(ADM), mRNA./ PROD = adrenomedullin/ FL = gb: NM001124.1 gb: D14874.1AF280545 Homo NRP2 −0.29 0.33 1.40 0.30 −1.67 0.61 1.22 0.33 sapiensneuropilin- 2b(5) (NRP2) mRNA, complete cds, alternatively spliced./PROD = neuropilin- 2b(5)/FL = gb: AF280544.1 gb: AF280545.1 NM_014624Homo S100A6 3.08 0.38 3.10 0.18 −0.57 0.37 3.24 0.19 sapiens 100calcium- binding protein A6 (calcyclin) (S100A6), mRNA./ PROD = S100calcium- binding protein A6/ FL = gb: NM_014624.2 gb: BC001431.1AB030824 Homo KLF5 0.57 0.24 2.16 0.49 −0.41 0.05 2.53 0.17 sapiens mRNAfor transcription factor BTEB2, complete cds./PROD = transcriptionfactor BTEB2/FL = gb: AF132818.1 gb: AF287272.1 gb: AB030824.1 gb:NM_001730.1 gb: D14520.1 NM_015675 Homo GADD45B 2.47 0.22 4.44 0.46 2.020.09 4.64 0.33 sapiens growth arrest and DNA- damage- inducible, beta(GADD45B), mRNA./PROD = DKFZP566B133 protein FL = gb: NM_015675.1 gb:AF090950.1 BF347089 tissue TIMP3 0.57 0.13 2.01 0.29 −0.19 0.20 2.090.44 inhibitor of metalloproteinase 3 (Sorsby fundus dystrophy,pseudoinflammatory)/ FL = gb: NM_000362.2 gb: U14394.1 gb: U67195.1 gb:U02571.1 BF056473 ESTs — −0.64 0.87 1.79 0.10 −0.73 0.34 1.57 0.23AA809487 Homo — 0.52 0.22 3.68 0.43 1.39 0.22 3.89 0.48 sapiens cDNA:FLJ21715 fis, clone COL10287, highly similar to AF071569 Homo sapiensmultifunctional calciumcalmodulin- dependent protein kinase II delta2isoform mRNA AL575735 collagen, — 5.54 0.11 6.65 0.14 4.57 0.08 6.680.07 type V, alpha 2/ FL = gb: NM_000393.1 AF003934 Homo GDF15 2.74 0.204.01 0.41 0.72 0.17 4.34 0.50 sapiens prostate differentiation factormRNA, complete cds./PROD = prostate differentiation factor/FL = gb:U88323.1 gb: BC000529.1 gb: AF003934.1 gb: NM_004864.1 gb: AF019770.1gb: AB000584.1 NM_000313 Homo PROS1 −1.54 0.10 0.98 0.83 −0.62 0.09 1.790.86 sapiens protein S (alpha) (PROS1), mRNA./PROD = protein S (alpha)/FL = gb: M15036.1 gb: NM_000313.1 NM_016651 Homo DACT1 2.37 0.26 4.790.67 2.46 0.14 3.92 0.78 sapiens heptacellular carcinoma novel gene-3protein (LOC51339), mRNA./PROD = heptacellular carcinoma novel gene-3protein/FL = gb: NM_016651.2 gb: AF251079.2 NM_020129 Homo LGALS14 1.380.30 3.15 0.70 0.79 0.04 2.47 0.66 sapiens placental protein 13- likeprotein (LOC56891), mRNA./PROD = placental protein 13-like protein/FL =gb: NM_020129.1 gb: AF267852.1 NM_013451 Homo FER1L3 1.75 0.15 4.02 0.971.59 0.13 5.14 0.82 sapiens fer-1 (C.elegans)- like 3 (myoferlin)(FER1L3), mRNA./ PROD = fer-1 (C.elegans)- like 3 (myoferlin)/ FL = gb:NM_013451.1 gb: AF182316.1 R72286 microfibrillar- MFAP4 −1.15 0.60 −1.760.61 −2.37 0.22 −0.60 0.72 associated protein 4 AI417362 ESTs, FUT1 2.420.14 2.32 0.72 −0.46 0.53 1.70 0.59 Moderately similar to ALU1_HUMAN ALUSUBFAMILY J SEQUENCE CONTAMINATION WARNING ENTRY (H.sapiens) NM_001553Homo apien IGFBP7 3.41 0.19 4.63 1.07 1.65 0.10 3.34 1.16 insulin-likegrowth factor binding protein 7 (IGFBP7), mRNA./ PROD = insulin- likegrowth factor binding protein 7/ FL = gb: NM_001553.1 gb: L19182.1BG285011 Homo ARID5B 0.05 0.22 1.79 0.21 −0.95 0.42 1.16 0.55 sapiensmRNA; cDNA DKFZp586N012 (from clone DKFZp586N012) BE967311 Homo — 1.990.06 4.08 0.92 2.28 0.17 5.01 0.87 sapiens mRNA; CDNA DKFZp762O1615(from clone DKFZp762O1615) BC005047 Homo DUSP6 2.50 0.32 4.04 0.67 1.560.16 2.94 0.84 sapiens, clone MGC: 12852, mRNA, complete cds./ PROD =Unknown (protein for MGC: 12852)/ FL = gb: NM_001946.1 gb: AB013382.1 b:BC003562.1 gb: BC003143.1 gb: BC005047.1 AW005572 putative 47 ANKS1B−0.59 0.12 0.19 0.70 −1.59 0.07 0.86 0.92 kDa protein AW294092 ESTs RERG0.54 0.16 −0.68 1.55 −2.78 0.92 −0.58 0.97 NM_001899 Homo CST4 0.14 0.522.46 1.59 0.53 0.14 3.87 1.40 sapiens cystatin S (CST4), mRNA./ PROD =cystatin S/ FL = gb: NM_001899.1 AI917371 ESTs — −1.47 0.95 0.18 1.43−1.24 0.33 2.21 1.17 NM_000515 Homo CSH1 −2.36 0.29 1.92 0.10 −0.73 0.571.37 0.21 sapiens growth hormone 1 (GH1), transcript variant 1,mRNA./PROD = growth hormone 1, isoform 1 precursor/ FL = gb: NM_000515.2NM_004414 Homo DSCR1 2.14 0.03 3.85 0.26 1.74 0.16 3.34 0.36 sapiensDown syndrome critical region gene 1 (DSCR1), mRNA./ PROD = Downsyndrome critical region protein 1/ FL = gb: U28833.2 gb: NM_00444.2AI355441 sprouty — 0.24 0.48 .72 0.50 −1.12 0.40 2.03 0.6 (Drosophila)homolog 4 AB032953 Homo ODZ2 0.00 0.14 0.65 0.35 −1.74 0.74 0.88 0.03sapiens mRNA for KIAA1127 protein, partial cds./ PROD = KIAA1127 proteinBE048571 ESTs MGC16121 −1.66 0.78 1.44 1.00 −1.31 0.21 0.10 1.22AW471145 ESTs PRSS23 0.87 0.10 3.46 0.35 .13 0.11 3.37 0.53 BF196943ESTs USP53 1.43 0.03 3.24 0.92 0.85 0.26 2.32 0.88 AF498927 Homo ARHGDIB−0.03 0.19 0.04 0.96 −3.00 0.54 0.85 0.93 sapiens Rho GDP dissociationinhibitor beta (ARHGDIB) mRNA, complete cds./ PROD = Rho GDPdissociation inhibitor beta/ FL = gb: AF498927.1 AF329092 Homo sapiensDOC1 −2.38 0.45 1.00 0.19 −1.62 0.74 0.75 0.11 GPBP-interacting protein90 mRNA, complete cds./ PROD = GPBP- interacting protein 90/ FL = gb:AF329092.1 BG250721 Homo sapiens — 2.48 0.04 4.54 0.95 2.63 0.14 5.310.83 mRNA; cDNA DKFZp564C2063 (from clone DKFZp564C2063 N690912063) ESTsPCDH17 0.59 0.10 1.49 0.88 −1.11 0.69 2.49 0.78 BF589359 ESTs PAG1 −1.110.52 −0.09 0.43 −1.54 0.12 0.50 0.13 BF968270 ESTs SLC35F3 0.37 0.021.84 0.30 0.10 0.17 2.46 0.36 NM_006183 Homo NTS 4.77 0.15 4.73 1.453.01 0.19 3.21 1.47 sapiens neurotensin (NTS), mRNA./ PROD = neurotensinprecursor/FL = gb: NM_006183.2 gb: U91618.1 D28124 Cluster Incl. NBL12.90 0.03 4.24 0.37 2.09 0.08 4.44 0.38 D28124: Human mRNA foru nknownproduct, complete cds/ cds = (61,603)/ gb = D28124/ gi = 641821/ ug =Hs.76307/ len = 1929 AW129593 tudor repeat TDRD7 1.19 0.19 2.40 0.870.92 0.04 3.27 0.89 associator with PCTAIRE 2 BE675435 core KLF6 0.290.30 2.91 1.02 0.54 0.24 3.68 0.94 promoter element binding protein/ FL= gb: AF001461.1 gb: BC000311.1 gb: NM_001300.2 gb: AB017493.1 b:BC004301.1 AI202327 ESTs CPEB2 1.88 0.09 3.42 0.07 0.99 0.10 3.36 0.03NM_002228 Homo JUN 2.12 0.16 4.09 0.56 1.62 0.13 4.53 0.41 sapiens v-junavian sarcoma virus 17 oncogene homolog (JUN), mRNA./ PROD = v-jun aviansarcoma virus 17 oncogene homolog/ FL = gb: NM_002228.2 gb: BC002646.1AF005775 Homo sapiens CFLAR 2.88 0.18 3.70 1.56 0.35 0.17 5.22 1.69caspase-like apoptosis regulatory protein 2 (clarp) mRNA, alternativelyspliced, complete cds./PROD = caspase-like apoptosis regulatory protein2/ FL = gb: AF005775.1 NM_007173 Homo sapiens PRSS23 3.02 0.10 5.11 0.212.90 0.05 5.22 0.23 protease, serine 23 (SPUVE), mRNA./PROD = protease,serine, 23/FL = gb: BC001278.1 gb: AF193611.1 gb: AF015287.1 gb:AL136914.1 gb: NM_007173.1 NM_012413 Homo QPCT 1.44 0.09 0.97 0.85 −2.460.65 0.19 1.10 sapiens glutaminyl- peptide cyclotransferase (glutaminylcyclase) (QPCT), mRNA./ PROD = glutaminyl- peptide cyclotransferaseprecursor/FL = gb: NM_012413.2 BU683415 Homo KLF6 3.40 0.06 5.28 0.853.46 0.09 6.01 0.85 sapiens, clone IMAGE: 4096 273, mRNA AV729634 DnaJDNAJC6 0.62 0.20 2.59 0.79 0.99 0.19 3.35 0.65 (Hsp40) homolog,subfamily B, member 6/FL = gb: AB007942.1 gb: NM_014787.1 BC038556 Homo— −0.79 0.40 0.12 0.99 −2.17 0.62 −0.08 0.38 sapiens, clone IMAGE: 3446976, mRNA. NM_014942 Homo ANKRD6 0.62 0.04 2.1 1.11 0.59 0.23 3.30 1.0sapiens KIAA0957 protein (KIAA0957), mRNA./ PROD = KIAA0957 protein/FL =gb: AB023174.1 gb: NM_014942.1 AF260333 Homo C4orf18 −1.58 0.10 0.211.21 −3.19 1.07 1.60 1.12 sapiens AD036 mRNA, complete cds./PROD =AD036/ FL = gb: AF260333.1 AA448956 Homo CAMK2D 1.47 0.18 2.75 0.42 0.210.21 2.97 0.39 sapiens cDNA: FLJ21715 fis, clone COL10287, highlysimilar to AF071569 Homo sapiens multifunctional calcium- calmodulin-dependent protein kinase Il delta2 isoform mRNA/ FL = gb: AF071569.1BE349115 ESTs COL22A1 −0.02 0.52 2.04 0.25 0.24 0.06 2.38 0.22 BF209337Homo LOC541471 4.83 0.17 5.80 0.73 3.13 0.02 4.84 0.81 apien CDNAFLJ10934 fis, clone OVARC1000640 AB019695 Homo — 2.43 0.01 4.15 0.401.42 0.05 3.50 0.29 sapiens mRNA for thioredoxin reductase II beta,complete cds./PROD = thioredoxin reductase II beta/FL = gb: AB019695.1AK090497 Homo LOC284576 −3.78 0.56 −2.87 1.42 −4.94 0.07 −4.74 0.53sapiens cDNA FLJ33178 fis, clone ADRGL2002753. NM_006763 Homo BTG2 2.300.10 3.28 0.62 0.85 0.25 3.84 0.74 sapiens BTG family, member 2 (BTG2),mRNA./PROD = BTG family, member 2/ FL = gb: U72649.1 gb: NM_006763.1BC002616 Homo TAGLN2 5.61 0.20 5.56 0.17 2.91 0.18 5.40 0.12 sapiens,transgelin 2, clone MGC: 2989, mRNA, complete cds. PROD = transgelin 2/FL = gb: BC002616.1 AF078077 Homo GADD45B 1.30 0.17 3.41 0.31 0.61 0.123.05 0.29 sapiens growth arrest and DNA- damage- inducible proteinGADD45bet a mRNA, complete cds./ PROD = growth arrest and DNA- damage-inducible proteinGAD D45beta/ FL = gb: AF087853.1 gb: AF078077.1NM_001854 Homo COL11A1 2.90 0.02 4.06 1.44 1.87 0.06 2.68 1.43 sapienscollagen, type XI, alpha 1 (COL11A1), mRNA./PROD = collagen, type XI,alpha 1/ FL = gb: J04177.1 gb: NM_001854.1 AI830201 ESTs KIAA0773 −0.750.82 1.24 0.22 −1.87 0.51 0.40 0.22 N95437 ESTs LMCD1 0.74 0.12 2.720.57 0.39 0.14 3.05 0.42 BC002511 Homo CBR1 3.72 0.02 1.03 2.43 −4.100.16 −1.64 2.36 sapiens, carbonyl reductase 1 clone MGC: 1920, RNA,complete cds,/ PROD = carbonyl reductase 1/ FL = gb: BC002511.1 gb:NM_001757.1 gb: J04056.1 AV682252 HIV-1 rev — −0.33 0.15 1.78 1.36 −0.240.17 0.41 1.29 binding protein 2 AW263497 ESTs SYTL5 −1.05 0.25 1.440.38 −0.55 0.41 2.11 0.47 AF130095 Homo — 5.80 0.14 6.80 0.49 4.54 0.107.35 0.36 sapiens clone FLC0562 PRO2841 mRNA, complete cds./PROD =PRO2841/ FL = gb: AF130095.1 H92988 tyrosine 3- C9orf19 2.00 0.06 3.540.91 1.93 0.14 4.61 0.81 monooxygen asetryptophan 5-monooxygenaseactivation protein, eta polypeptide X02761 Human FN1 5.67 0.17 6.65 0.544.52 0.21 7.21 0.42 mRNA for fibronectin (FN precursor)./ PROD =fibronectin precursor AI016316 ESTs — 0.24 0.18 1.19 1.17 −0.33 0.180.25 1.18 NM_006622 Homo PLK2 4.64 0.14 5.88 0.45 3.50 0.06 5.19 0.47sapiens serum- ind cible kinase (SNK), mRNA./ PROD = serum- induciblekinase/ FL = gb: AF059617.1 gb: NM_006622.1 gb: AF223574.1 NM_013238Homo DNAJC15 −2.27 0.60 3.79 2.03 4.07 0.09 5.58 2.31 sapiens DNAJdomain- containing (MCJ), mRNA./ PROD = DNAJ domain- containing/ FL =gb: NM_013238.1 gb: AF126743.1 AK026737 Homo FN1 5.86 0.14 6.85 0.494.66 0.08 7.39 0.37 sapiens CDNA: FLJ23084 fis, clone LNG06602, highlysimilar to HSFIB1 Human mRNA for fibronectin (FN precursor). NM_001458Homo FLNC 3.28 0.17 4.17 0.70 2.11 0.17 3.39 0.64 sapiens filamin C,gamma (actin- binding protein-280) (FLNC), RNA./ PROD = gamma filamin/FL = gb: AF089841.1 gb: NM_001458.1 AK025843 Homo PALLD 1.58 0.31 3.380.15 1.03 0.20 3.29 0.19 sapiens cDNA: FLJ22190 fis, clone HRC01053./ FL= gb: AF151909.1 gb: AF077041.1 gb: NM_016081.1 BC005858 Homo FN1 5.860.10 6.86 0.49 4.70 0.19 7.37 0.36 sapiens, clone MGC: 3255, mRNA,complete cds./ PROD = Unknown (protein for MGC: 3255)/ FL = gb:BC005858.1 BG491844 v-jun avian JUN 3.53 0.08 5.45 0.47 3.42 0.11 5.910.34 sarcoma virus 17 oncogene homolog/FL = gb: NM_002228.2 gb:BC002646.1 AA284532 tyrosine 3- C9orf19 2.19 0.11 4.12 0.84 2.43 0.084.93 0.90 monooxygen asetryptophan 5- monooxygenase activation rotein,eta polypeptide AA192306 triadin/FL = TRDN −2.54 0.69 0.02 0.61 −1.990.31 0.57 0.63 gb: U18985.1 gb: NM_006073.1 AF116676 Homo — 1.50 0.183.31 1.11 1.37 0.15 4.23 1.05 sapiens PRO1957 mRNA, complete cds./ PROD= PRO1957/ FL = gb: AF116676.1 NM_003033 Homo ST3GAL1 −0.08 0.18 1.681.04 0.20 0.16 2.74 0.74 sapiens sialyltransferase 4A (beta-galactosidase alpha-2,3- sialytransferase) (SIAT4A), mRNA./ PROD =sialyltransferase 4A (beta- galactosidasealpha- 2,3-sialytransferase)/FL = gb: L13972.1 gb: L29555.1 gb: NM_003033.1 AI222435 ESTs — 4.14 1.52−0.73 0.32 −2.33 0.62 −0.33 0.12 NM_001924 Homo GADD45A 3.59 0.09 5.040.44 3.06 0.17 5.38 0.48 sapiens growth arrest and DNA- damage-inducible, alpha GADD45A), mRNA./ PROD = growth arrest and DNA- damage-inducible, alpha/ FL = gb: M60974.1 gb: NM_001924.2 NM_001425 Homo EMP32.76 0.18 3.90 0.64 1.51 0.06 3.13 0.57 sapiens epithelial membraneprotein 3 (EMP3), mRNA./ PROD = epithelial membrane protein 3/ FL = gb:U52101.1 gb: NM_001425.1 gb: U87947.1 AB017493 Homo KLF6 1.69 0.20 4.021.01 1.80 0.08 4.87 0.87 sapiens mRNA for DNA-binding zinc finger(GBF),complete cds./ PROD = DNA- binding zinc finger(GBF)/ FL = gb: AF001461.1gb: BC000311.1 gb: NM_001300.2 gb: AB017493.1 gb: BC00430.1 X58851 Human— 0.99 0.16 2.75 .28 1.12 0.15 4.04 .14 MLC1emb gene for embryonicmyosin alkaline light chain, promoter and exon 1 BE327172 v-jun avian —0.86 0.15 2.94 0.51 1.14 0.08 3.57 0.45 sarcoma virus 17 oncogenehomolog U37283 Human MFAP5 0.48 0.19 1.87 0.26 0.15 0.35 1.36 0.47microfibril- associated glycoprotein- 2 MAGP-2 mRNA, complete cds./ PROD= microfibril- associated glycoprotein- 2 MAGP-2/ FL = gb: NM_003480.1gb: U37283.1 AI819043 ESTs CREB5 0.92 0.26 2.63 0.73 0.65 0.15 1.80 0.79NM_001511 Homo CXCL1 1.01 0.13 2.95 0.35 0.31 0.08 2.39 0.27 sapiensGRO1 oncogene (melanoma growth stimulating activity, alpha) (GRO1),mRNA./ PROD = GRO1 oncogene (melanoma growth timulating- activity,alpha)/ FL = gb: NM_001511.1 NM_006736 Homo DNAJB2 2.23 0.04 3.46 0.441.10 0.02 3.75 0.41 sapiens heat shock protein, neuronal DNAJ-like 1(HSJ1), mRNA./ PROD = heat shock protein, neuronal DNAJ-like 1/ FL = gb:NM_006736.1 AA534817 ESTs, EDG3 2.32 0.06 3.28 1.04 2.08 0.04 4.58 1.04Weakly similar to ALU8_HUMAN ALU SUBFAMILY SX SEQUENCE CONTAMIN- ATIONWARNING ENTRY (H.sapiens) U82164 Human CD99 4.73 0.15 5.69 0.73 3.190.07 6.36 0.93 transmembrane protein CD99 type II mRNA, complete cds./PROD = CD99 typeII/ FL = gb: BC002584.1 gb: NM_002414.1 gb: M16279.1 gb:BC003147.1 b: U82164.1 NM_000389 Homo CDKN1A 4.03 0.02 4.20 0.12 1.810.07 4.16 0.05 b: U82164.1 sapiens cyclin- dependent kinase inhibitor 1A(p21, Cip1) (CDKN1A), mRNA./ PROD = cyclin- dependent kinase inhibitor1A (p21,Cip1)/ FL = gb: U03106.1 gb: BC000275.1 gb: BC001935.1 gb:L25610.1 gb: U09579.1 gb: NM_000389.1 gb: L26165.1 NM_001299 Homo CNN13.75 0.15 5.75 0.50 3.47 0.11 5.14 0.43 sapiens calponin 1, basic,smooth muscle (CNN1), mRNA./ PROD = calponin 1, basic, smooth muscle/ FL= gb: U37019.1 gb: NM_001299.1 gb: D17408.1 M36172 Human MYL4 1.55 0.213.30 1.05 1.21 0.13 4.11 1.06 embryonic myosin alkali light chain (MLC1)RNA, complete cds./ FL = gb: M36172.1 gb: M24121.1 gb: NM_002476.1AB033831 Homo PDGFC 0.48 0.21 0.63 0.15 −1.87 0.96 0.21 0.16 sapienshSCDGF mRNA for spinal cord- derived growth factor, complete cds./ PROD= spinal cord- derived growth factor/ FL = gb: NM_016205.1 gb:AB033831.1 gb: AF091434.1 gb: AF244813.1 NM_014333 Homo IGSF4 3.11 0.094.12 0.27 1.85 0.06 3.79 0.24 sapiens immunoglobulin superfamily, member4 (IGSF4), mRNA./ PROD = immunoglobulin superfamily, member 4/ FL = gb:NM_014333.1 gb: AF132811.1 AF345910 Homo TTC29 0.35 0.26 1.53 0.93 −0.510.52 0.91 0.72 sapiens YD-SP14 mRNA, complete cds./ PROD = NYD- SP14/ FL= gb: AF345910.1 M_004297 Homo GNA14 4.59 0.06 4.52 1.59 1.95 0.11 2.951.50 sapiens guanine nucleotide binding protein (G protein), alpha 14(GNA14), mRNA./ PROD = guanine nucleotide binding protein (G protein),alpha 14/ FL = gb: AF105201.1 gb: NM_004297.1 AK057525 Homo — 3.60 0.084.45 0.44 2.00 0.16 4.80 0.46 sapiens CDNA FLJ32963 fis, cloneTESTI2008405. BC000893 Homo HIST1H2BK 2.80 0.06 3.99 0.90 2.48 0.09 4.810.82 sapiens, H2B histone family, member A, clone MGC: 5132, mRNA,complete cds./PROD = H2B histone family, member A/ FL = gb: BC000893.1NM_007038 Homo ADAMTS5 0.23 0.17 1.80 0.48 0.45 0.28 1.11 0.73 sapiens adisintegrin- like and metalloprotease (reprolysin type) withthrombospondin type 1 motif, 5 (aggrecanase-2) (ADAMTS5), mRNA./ PROD =adisintegrin and metalloprotease withthrombospondin motifs-5preproprotein/ FL = gb: NM_007038.1 gb: AF14209 AW241910 ESTs, COL22A1−0.21 0.12 1.15 0.47 −0.57 0.18 1.90 0.51 Weakly similar to JX0369collagen alpha 1(XIX) chain precursor (H.sapiens) AI860150 ESTs, FOSL2−0.43 0.55 1.59 0.37 −0.34 0.10 1.08 0.20 Weakly similar to A49134 Igkappa chain V-I region (H.sapiens) M_005902 Homo SMAD3 1.09 0.41 1.490.25 −1.10 0.89 1.73 0.10 sapiens MAD (mothers against decapentaplegic,Drosophila) homolog 3 (MADH3), mRNA./ PROD = MAD (mothers againstdecapentaplegic, Drosophila) homolog 3/ FL = gb: U68019.1 gb: U76622.1gb: NM_005902.1 AA777512 Homo CAMK2D 2.27 0.15 3.67 0.47 1.67 0.08 3.990.51 sapiens cDNA: FLJ21715 fis, clone COL10287, highly similar toAF071569 Homo sapiens multifunctional calcium- calmodulin- dependentprotein kinase II delta2 isoform mRNA AI130705 ESTs, FAM89A 0.80 0.001.96 0.99 0.43 0.18 2.70 0.97 Weakly similar to A46302 PTB-ssociatedsplicing factor, long form (H.sapiens) NM_007061 Homo CDC42EP1 1.86 0.112.51 0.29 0.16 0.27 2.00 0.17 sapiens serum constituent protein (MSE55),mRNA./ PROD = serum constituent protein/ FL = gb: M88338.1 gb:NM_007061.1 NM_003407 Homo ZFP36 2.55 0.14 3.53 0.55 1.49 0.28 3.98 0.47sapiens zinc finger protein homologous to Zfp-36 in mouse (ZFP36),mRNA./ PROD = zinc finger protein homologous to Zfp-36 inmouse/ FL = gb:NM_003407.1 gb: M92843.1 gb: M63625.1 BC033088 Homo LMNA 2.03 0.22 2.890.45 0.69 0.24 1.79 0.50 sapiens, Similar to lamin AC, clone MGC: 45654IMAGE: 3623265, mRNA, complete cds./ PROD = Similr to lamin AC/ FL = gb:BC033088.1 U97075 Homo CFLAR 2.50 0.09 3.39 1.45 0.73 0.08 4.93 1.54sapiens FLICE-like inhibitory protein short form mRNA, complete cds./PROD = FLICE- like inhibitory protein short form/FL = gb: U97075.1AF133207 Homo HSPB8 2.54 0.07 4.47 0.62 2.22 0.04 4.88 0.58 sapiensprotein kinase (H11) mRNA, complete cds./PROD = protein kinase/ FL = gb:AF133207.1 NM_005979 Homo S100A13 4.10 0.11 5.67 1.17 3.92 0.02 6.811.20 sapiens S100 calcium- binding protein A13 (S100A13), mRNA./ PROD =S100 calcium- binding protein A13/ FL = gb: BC000632.1 gb: NM_005979.1AL040178 ESTs PEAR1 −0.87 0.08 0.51 0.36 −1.16 0.30 0.48 0.26 AL117523Homo SAMD4A 0.47 0.06 1.59 0.61 −0.24 0.30 1.28 0.53 apien mRNA; cDNADKFZp434H0350 (from clone DKFZp434H0350); partial cds./ PROD =hypothetical protein AB051826 Homo RHOU 0.55 0.23 2.18 0.61 0.17 0.292.73 0.65 sapiens hG28K mRNA for GTP-binding protein like 1, completecds./PROD = GTP-binding protein like 1/ FL = gb: AF282258.1 gb:NM_021205.1 gb: AB051826.1 BC005961 Homo PTHLH −3.33 0.69 −0.88 0.81−2.86 0.77 −1.97 1.36 sapiens, parathyroid hormone-like hormone, cloneMGC: 14611, mRNA, complete cds./ PROD = para thyroid hormone-likehormone/ FL = gb: BC005961.1 AI670948 ESTs NODAL 2.13 0.04 2.37 0.220.42 0.13 2.78 0.38 AI685060 caldesmon 1/ CALD1 4.34 0.33 6.42 0.73 4.450.09 5.63 0.82 FL = gb: M6410.1 gb: NM_004342.2 BF797381 Homo CAMK2D3.16 0.13 4.9 0.65 3.10 0.10 5.49 0.6 sapiens CDNA: FLJ21715 fis, cloneCOL10287, highly similar to AF071569 Homo sapiens multifunctionalcalcium- calmodulin- dependent protein kinase II delta2 isoform mRNAAF026219 Homo DLC1 1.16 0.07 2.28 1.13 0.61 0.09 3.52 0.84 sapiens HPprotein (HP) mRNA, complete cds./PROD = HP protein/FL = gb: AF026219.1gb: AF035119.1 gb: NM_006094.2 AK024480 Homo LOC126917 1.64 0.11 2.860.24 .19 0.06 2.58 0.25 sapiens mRNA for FLJ00074 protein, partial cds./PROD = FLJ00074 protein N29837 ESTs LIX1 −1.43 0.15 −0.11 0.33 −1.500.45 −0.22 0.22 AK001022 Homo ISL2 0.41 0.34 2.14 0.39 0.32 0.08 1.570.49 apien cDNA FLJ10160 fis, clone HEMBA1003545, highly similar toINSULIN GENE ENHANCER PROTEIN ISL-2. NM_000047 Homo ARSE 1.37 0.11 2.451.03 0.59 0.05 3.18 1.23 sapiens arylsulfatase E (chondrodysplasiapunctata 1) (ARSE), mRNA./PROD = arylsulfatase E precursor/FL = gb:X83573.1 gb: NM_000047.1 NM_006379 Homo SEMA3C 0.24 0.25 1.08 0.00 −0.340.05 0.86 0.18 sapiens sema domain, immunoglobulin domain (lg), shortbasic domain, secreted, (semaphorin) 3C (SEMA3C), mRNA./ PROD = semadomain, immunoglobulin domain (lg), shortbasic domain, secreted,(semaphorin) 3C/ FL = gb: NM_006379.1 gb: AB000220.1 NM_007127 Homo VIL10.31 0.30 1.92 0.49 0.02 0.22 2.54 0.68 sapiens villin 1 (VIL1), mRNA./PROD = villin 1/ FL=gb: NM_007127.1 U76549 Human KRT8 5.41 0.16 5.870.56 3.79 0.05 6.34 0.57 cytokeratin 8 mRNA, complete cds./PROD =cytokeratin 8/ FL = gb: BC000654.1 gb: U76549.1 gb: NM_002273.1 gb:M26324.1 gb: M34225.1 NM_004904 Homo CREB5 0.92 0.23 1.84 0.37 0.00 0.061.21 0.80 sapiens cAMP response element- binding protein CRE- BPa(H_GS165L1 5.1), mRNA./ PROD = cAMP response element- binding proteinCRE-BPa/ FL = gb: NM_004904.1 b: L05911.1 AW082836 ESTs, WNK4 −1.17 0.260.98 0.38 −0.97 0.20 0.05 0.86 Weakly similar to B34087 hypotheticalprotein (H.sapiens) BE568134 death TNFRSF21 4.76 0.06 5.90 0.54 3.940.08 6.28 0.58 receptor 6/FL = gb: NM_014452.1 gb: AF068868.1 NM_002845Homo PTPRM 2.50 0.10 3.43 0.46 1.34 0.19 3.76 0.50 sapiens proteintyrosine phosphatase, receptor type, M (PTPRM), mRNA./ PROD = proteintyrosine phosphatase, receptor type, mupolypeptide/ FL = gb: NM_002845.1AI949419 ESTs — −0.11 0.21 1.72 0.64 −0.47 0.01 2.39 0.77 AK024680 HomoNRP2 0.42 0.02 2.65 0.17 0.98 0.09 2.86 0.17 sapiens cDNA: FLJ21027 fis,clone CAE07110./ FL = gb: NM_018534.1 BE542563 ESTs LOC643277 2.20 0.100.54 1.49 −3.73 0.40 −1.74 1.96 AW005572 putative 47 ANKS1B −1.21 0.52−0.24 0.82 −1.19 0.55 1.05 0.64 kDa protein AW665892 paternally MFAP5−3.87 0.82 −1.56 1.24 −2.40 0.24 −3.49 2.10 expressed 3 NM_006206 HomoPDGFRA 0.92 0.18 2.34 0.74 0.72 0.09 3.18 0.62 sapiens latelet- derivedgrowth factor receptor, alpha polypeptide (PDGFRA), mRNA./ PROD =platelet- derived growth factor receptor, alphapolypeptide/ FL = gb:NM_006206.1 gb: M21574.1 NM_002425 Homo MMP10 −1.21 0.70 1.21 0.88 −0.370.28 0.52 0.45 sapiens matrix metalloproteinase 10 (stromelysin 2)(MMP10), mRNA./ PROD = matrix metalloproteinase 10 preproprotein/ FL =gb: BC002591.1 gb: NM_002425.1 NM_004338 Homo C18orf1 −0.79 0.22 −0.090.39 −1.57 0.43 0.50 0.02 sapiens chromosome 18 open reading frame 1(C18ORF1), mRNA./ PROD = chromosome 18 open reading frame 1/ FL = gb:NM_4338.1 gb: AF009426.1 AF 52094 Homo EPAS1 .75 .12 2.28 .19 0.42 .211.74 .33 sapiens clone 23698 mRNA sequence./ FL = gb: U51626.1 gb:U81984.1 gb: NM_001430.1 BF126155 ESTs S100A10 −0.32 0.25 1.24 0.28−0.65 .33 1.02 0.44 AI860212 phosphoprotein PAG1 −0.17 0.22 1.37 0.28−0.40 .23 1.25 0.13 associated with GEMs/ FL = gb: AF240634.1 gb:NM_018440.1 AL110298 Homo SLC2A14 5.13 0.06 6.03 0.58 3.97 .24 6.33 0.73sapiens mRNA; CDNA DKFZp564K1672 (from clone DKFZp564K1672); partialcds./ PROD = hypothetical protein AY048775 Homo MANEA −0.47 0.40 0.710.90 −0.69 .35 1.23 0.81 sapiens mandaselin long form mRNA, completecds./ PROD = mandaselin long form/ FL = gb: AY048775.1 M99436 ClusterIncl. TLE2 2.65 0.07 4.05 0.66 1.95 .13 4.76 0.61 M99436: Humantransducin-like enhancer protein (TLE2) mRNA, complete cds/ cds =(25,2256)/ gb = M99436/ gi = 307511/ ug = Hs.173063/ len = 2271NM_014061 Homo MAGEH1 2.0 0.23 3.8 1.17 1.08 0.24 4.86 1.12 sapiensAPR-1 protein (APR-1), mRNA./ PROD = APR-1 protein/ FL = gb: AF320912.1gb: AF143235.3 gb: NM_014061.1 AL577531 caldesmon 1/ CALD1 5.79 0.066.06 0.75 .07 0.06 5.13 0.77 FL = gb: M64110.1 gb: NM_004342.2 AI082237proprotein TAGLN 1.38 0.21 3.20 0.33 1.55 0.15 2.74 0.44 convertasesubtilisinkexin type 7 BF055171 acyl- ACOX3 0.88 0.22 2.52 0.83 1.270.06 3.73 0.81 Coenzyme A oxidase 3, pristanoyl/ FL = gb: NM_003501.1AF231124 Homo SPOCK1 2.84 0.09 4.17 0.95 2.71 0.07 5.38 1.00 sapienstestican-1 mRNA, omplete cds./ PROD = testican-1/ FL = gb: NM_004598.1gb: AF231124.1 AA588092 ESTs SLC40A1 −1.53 0.23 −0.94 0.51 −2.42 0.330.27 0.27 AK094809 Homo RASGRF2 3.20 0.05 3.64 0.77 1.95 0.24 2.95 0.74sapiens cDNA FLJ37490 fis, clone BRAWH2014934, highly similar to GUANINENUCLEOTIDE RELEASING PROTEIN. NM_013959 Homo NRG1 1.50 0.20 3.22 0.681.34 0.13 3.78 0.75 sapiens neuregulin 1 (NRG1), transcript variantSMDF, mRNA./ PROD = neuregulin 1 isoform SMDF/ FL = gb: L41827.1 gb:NM_013959.1 NM_004887 Homo CXCL14 0.70 0.07 1.70 0.37 −0.15 0.11 2.360.63 sapiens small inducible cytokine subfamily B (Cys-X-Cys), member 14(BRAK) (SCYB14), RNA./ PROD = small inducible cytokine subfamilyB(Cys-X- Cys), member 14 (BRAK)/ FL = gb: AF144103.1 gb: AF106911.1 gb:AF073957.1 gb: BC003513.1 gb: NM_004887.1 T77995 Homo — −0.20 0.16 0.820.50 −1.70 0.24 1.17 0.06 sapiens cDNA FLJ13392 fis, clone PLACE1001 280NM_030971 Homo SFXN3 −0.43 0.59 −0.01 0.78 −1.92 0.17 −1.25 0.43 sapienssimilar to rat tricarboxylate carrier-like protein (BA108L7.2), mRNA./PROD = similar to rat tricarboxylate carrier- likeprotein/ FL = gb:NM_030971.1 H25097 KIAA1350 USP53 3.25 0.05 4.57 0.20 2.96 0.04 4.470.13 protein NM_004932 Homo CDH6 −3.58 1.16 −1.12 0.51 −2.14 0.63 −0.880.39 sapiens cadherin 6, type 2, K- cadherin fetal kidney) (CDH6),mRNA./ PROD = cadherin 6, type 2, K- cadherin (fetal kidney)/ FL = gb:D31784.1 gb: NM_004932.1 N21426 hypothetical SYTL2 2.41 0.09 3.44 1.221.70 0.06 2.02 1.21 protein FLJ20163 AA234096 KIAA0963 MGC16121 0.380.16 1.74 0.63 −0.13 0.04 1.10 0.65 protein AV734843 hypothetical OBFC2A0.62 0.06 1.62 0.79 0.09 0.02 1.25 0.73 protein FLJ22833 AL519710immunoglobulin IGSF4 3.86 0.02 4.95 0.26 3.22 0.12 4.66 0.27superfamily, member 4/ FL = gb: NM_014333.1 gb: AF132811.1 N32834 HIV-1rev — −0.28 0.36 1.58 0.97 −0.23 0.18 0.33 1.17 binding protein 2AF132811 Homo IGSF4 2.23 0.07 3.85 0.28 2.17 0.10 3.58 0.34 sapiensnectin-like protein 2 (NECL2) mRNA, complete cds./ PROD = nectin- likeprotein 2/ FL = gb: NM_014333.1 gb: AF132811.1 J04177 Cluster Incl.COL11A1 3.28 0.22 4.44 1.22 2.32 0.03 3.03 1.31 J04177: Hum n alph-1type XI collagen type XI collagen (COL11A1) mRNA, complete cds/ cds =(161,5581)/ gb = J04177/ gi = 179729/ ug = Hs.82772/ len = 6158 AI982754clusterin CLU 0.17 0.14 1.86 0.94 0.39 0.13 0.97 0.83 (complement lysisinhibitor, SP- 40,40, sulfated glycoprotein 2, testosterone- repressedprostate message 2, apolipoprotein J) NM_003501 Homo ACOX3 0.43 0.322.10 0.91 0.79 0.08 3.19 0.95 sapiens acyl- Coenzyme A oxidase 3,pristanoyl (ACOX3), mRNA./ PROD = acyl- Coenzyme A oxidase 3,pristanoyl/ FL = gb: NM_003501.1 AF144103 Homo CXCL14 1.69 0.13 2.160.55 0.40 0.21 2.73 0.69 sapiens NJAC protein (NJAC) mRNA, completecds./ PROD = NJA protein/ FL = gb: AF144103.1 gb: AF106911.1 gb:AF073957.1 gb: BC003513.1 gb: NM_004887.1 NM_005451 Homo PDLIM7 2.580.12 3.12 0.50 1.30 0.16 2.65 0.37 sapiens enigma (LIM domain protein)(ENIGMA), mRNA./ PROD = enigma protein/ FL = gb: BC001093.1 gb:NM_005451.2 gb: AF265209.1 NM_004472 Homo FOXD1 −0.85 0.80 2.04 0.550.71 0.23 1.70 0.74 sapiens forkhead box D1 (FOXD1), mRNA./ PROD = forkhead box D1/ FL = gb: U59832.1 gb: NM_004472.1 AF332197 Homo SIX2 0.360.24 0.72 0.12 −0.44 0.35 1.05 0.15 sapiens adult SIX2 (SIX2) mRNA,complete cds./ PROD = SIX2/ FL = gb: AF332197.1 gb: NM_016932.1 b:AF136940.1 AB046817 Homo SYTL2 2.88 0.17 4.09 1.09 2.50 0.05 3.12 1.03sapiens mRNA for KIAA1597 protein, partial cds./ PROD = KIAA1597 proteinAK093435 Homo FLJ36116 4.75 0.02 4.59 1.53 2.46 0.18 6.15 1.46 sapiensCDNA FLJ36116 fis, clone TESTI2022338. NM_004815 Homo ARHGAP29 2.18 0.023.18 0.82 1.69 0.03 4.15 0.69 sapiens PTPL1- associated RhoGAP 1(PARG1), mRNA./ PROD = PTP L1- associated RhoGAP 1/ FL = gb: U90920.1gb: NM_004815.1 BG028597 ESTs COL11A1 0.19 0.02 1.61 1.02 −0.11 0.380.48 1.09 AB019562 Homo SPP1 0.58 0.14 2.79 1.07 1.10 0.02 1.21 1.06sapiens mRNA expressed only in placental villi, clone SMAP41. NM_002346Homo LY6E 3.54 0.06 3.92 1.16 1.81 0.23 4.93 1.39 sapiens lymphocyteantigen 6 complex, locus E LY6E), mRNA./ PROD = lymphocyte antigen 6complex, locus E/ FL = gb: U42376.1 gb: NM_002346.1 gb: U56145.1BF589515 ESTs TMEM16D 0.63 0.24 1.94 0.85 0.90 0.20 1.47 0.75 AL037401nuclear NR2F2 −2.00 0.30 0.58 0.67 −2.19 0.13 0.82 0.66 receptorsubfamily 2, group F, member 2/ FL = gb: M64497.1 NM_000783 Homo CYP26A13.92 0.27 6.28 1.06 6.29 0.09 7.31 0.98 sapiens cytochrome P450,subfamily XXVIA, polypeptide 1 (CYP26A1), mRNA./ PROD = cytochrome P450,subfamily XXVIA, polypeptide 1/ FL = gb: NM_000783.1 gb: AF005418.1U16307 Human GLIPR1 −0.11 0.03 1.16 0.95 −0.46 0.77 0.35 0.77 gliomapathogenesis- related protein (GliPR) mRNA, omplete cds./ PROD = gliomapathogenesis- related protein/FL = gb: NM_006851.1 gb: U16307.1NM_001233 Homo CAV2 4.07 0.07 4.42 0.90 2.82 0.08 3.44 0.85 sapienscaveolin 2 (CAV2), mRNA./ PROD = caveolin 2/ FL = gb: AF035752.1 gb:BC005256.1 gb: NM_001233.1 AA211909 ESTs C20orf100 0.62 0.17 1.93 1.00−0.21 0.11 0.72 0.99 AK057525 Homo — 2.17 0.13 2.97 0.77 0.97 0.08 3.680.60 sapiens cDNA FLJ32963 fis, clone TESTI2008405. BF344237 Homo —−2.82 0.20 −0.92 0.64 −0.73 0.67 −1.25 1.03 sapiens mRNA; CDNADKFZp564N1116 (from clone DKFZp564N1116) NM_014481 Homo APEX2 2.76 0.153.18 0.80 1.57 0.11 3.91 0.93 sapiens apurini- capyrimidinicendonuclease (APEX nuclease)- like 2 protein APEXL2), mRNA./ PROD =apurini- capyrimidinic endonuclease (APEXnuclease)- like 2 protein/ FL =gb: AB049211.1 gb: NM_014481.1 gb: BC002959.1 gb: AB021260.1 gb:AF119046.1 AI912583 HIV-1 rev KRR1 1.10 0.23 2.68 1.04 1.31 0.06 1.531.01 binding protein 2 BI254089 Homo ADAMTS5 −1.40 0.26 −0.01 0.31 −1.710.64 −0.79 0.52 sapiens full length insert cDNA clone ZD50E03 BF197655caveolin 2/ CAV2 3.51 0.10 3.54 0.94 2.32 0.09 2.70 0.85 FL = gb:AF035752.1 gb: BC005256.1 gb: NM_001233.1 NM_001955 Homo EDN1 2.84 0.152.97 0.95 1.01 0.14 1.75 1.03 sapiens endothelin 1 (EDN1), mRNA./ PROD =endothelin 1/ FL = gb: NM_001955.1 NM_003319 Homo TTN 1.28 0.19 0.620.86 −0.92 0.42 2.00 0.55 sapiens titin (TTN), mRNA./ PROD = titin FL =gb: NM_003319.1 BE965029 Homo sapiens MICAL2 0.60 0.21 1.95 0.64 0.210.19 0.83 0.78 cDNA: FLJ22463 fis, clone HRC10126 AI452457 ESTs C1orf168−1.48 0.53 0.02 0.93 −1.87 0.07 1.05 0.94 AI733465 collagen, COL9A2 0.960.13 1.99 1.00 1.17 0.14 2.94 0.86 type IX, alpha 2/ FL = gb:NM_001852.1 NM_006103 Homo sapiens WFDC2 4.02 0.11 5.11 1.05 3.39 0.046.19 1.10 epididymis-specific whey-acidic protein type, four-disulfidecore; putative ovarian carcinoma marker (HE4), mRNA./PROD =epididymis-specific, whey-acidic protein type, four- disulfide core;putative ovarian carcinoma marker/ FL = gb: NM_00610 NM_017540 Homosapiens GALNT10 1.46 0.18 2.68 0.78 0.97 0.23 3.47 0.82 hypotheticalprotein DKFZp586H0623 DKFZp586H0623), mRNA./PROD = hypothetical proteinDKFZp586H0623/ FL = gb: NM_017540.1 W72527 phosphoserine SLC1A4 −0.650.09 0.38 0.80 −0.63 0.62 −0.72 0.09 aminotransferase NM_003468 HomoFZD5 1.35 0.26 3.43 1.21 1.45 0.10 4.43 1.16 sapiens frizzled(Drosophila) homolog 5 (FZD5), mRNA./ PROD = frizzled 5/ FL = gb:NM_003468.1 gb: U43318.1 H15920 ESTs, MRGPRF 2.34 0.18 3.62 1.02 2.110.11 2.87 0.84 Weakly similar to RTA RAT PROBABLE G PROTEIN- COUPLEDRECEPTOR RTA (R.norvegicus) U83508 Human ANGPT1 1.06 0.11 1.49 0.44−0.32 0.27 0.32 0.79 angiopoietin- 1 mRNA, complete cds./ PROD =angiopoietin- 1/FL = gb: NM_001146.1 gb: D13628.1 gb: U83508.1 F043179Homo PRSS1 2.42 0.12 3.07 0.81 1.71 0.10 2.30 0.60 sapiens T cellreceptor beta chain (TCRBV13S1- TCRBJ2S1) mRNA, complete cds./ PROD = Tcell receptor beta chain/ FL = gb: AF043179.1 AU157541 hypothetical —1.39 0.08 2.12 0.42 0.55 0.18 1.41 0.77 protein FLJ22833/ FL = gb:NM_022837.1 AF114264 Homo NEXN 1.37 0.21 2.86 0.51 1.72 0.16 1.96 0.76sapiens clone HH409 unknown mRNA./ PROD = unknown BE965029 Homo MICAL21.58 0.19 2.58 0.51 1.32 0.13 1.40 0.74 sapiens CDNA: FLJ22463 fis,clone HRC10126 AB028976 Homo SAMD4A 2.89 0.08 3.65 0.66 2.04 0.17 2.780.81 sapiens mRNA for KIAA1053 protein, partial cds./ PROD = KIA A1053protein AI670862 ESTs, FOSL2 0.17 0.21 2.05 0.57 0.45 0.16 1.18 0.65Weakly similar to A49134 lg kappa chain V-I region H.sapiens) L03203Human PMP22 0.37 0.24 1.95 0.84 0.31 0.26 2.77 0.91 peripheral myelinprotein 22 (GAS3) mRNA, complete cds./ PROD = peripheral myelin protein22/ FL = gb: L03203.1 AI571798 Rho GDP ARHGDIA 0.89 0.06 −0.54 0.26−3.00 0.89 −2.58 0.87 dissociation inhibitor (GDI) alpha X57348 ClusterIncl. — 2.94 0.04 4.07 0.41 2.70 0.08 3.69 0.41 X57348: H.sapiens mRNA(clone 9112)/ cds = (165,911)/ gb = X57348/ gi = 23939/ ug = Hs.184510/len = 1407 AF051851 Homo SVIL 2.03 0.18 2.28 0.62 1.13 0.20 1.71 0.69sapiens supervillin mRNA, complete cds./ PROD = supervillin/ FL = gb:AF051851.1 gb: NM_003174.2 gb: AF051850.1 M95929 Human — 0.07 0.43 1.270.58 −0.02 0.19 0.64 0.61 homeobox protein (PHOX1) mRNA, 3 end./ PROD =homeobox protein BG251266 FOS-like FOSL1 1.94 0.06 2.41 0.46 1.25 0.051.93 0.36 antigen-1/ FL = gb: NM_005438.1 AW298375 ESTs I −0.40 0.300.46 0.63 −0.09 0.20 −0.58 1.07 NM_004362 Homo CLGN 0.90 0.30 1.95 0.960.69 0.11 3.11 1.01 sapiens calmegin (CLGN), mRNA./ PROD = calmegin/ FL= gb: NM_004362.1 gb: D86322.1 AF001540 calcineurin- — 0.10 0.15 2.840.70 0.42 0.44 1.64 0.39 binding protein calsarcin-1 NM_001191 HomoBCL2L1 1.21 0.22 0.58 0.35 −1.89 0.35 −1.09 1.03 sapiens BCL2-like 1(BCL2L1), mRNA./ PROD = BCL2- like 1/FL = gb: NM_001191.1 NM_003316 HomoTTC3 3.90 0.15 5.15 0.98 3.68 0.12 6.19 0.97 sapiens tetratricopeptiderepeat domain 3 (TTC3), mRNA./ PROD = tetratricopeptide repeat domain 3/FL = gb: D84295.1 gb: NM_003316.1 L16895 Human lysyl — −0.66 0.24 0.530.14 −0.63 0.15 0.14 0.19 oxidase (LOX) gene, exon 7 AI912976 ESTsRASGRF2 3.13 0.08 4.05 0.56 2.66 0.16 3.54 0.64 NM_012242 Homo DKK1 0.470.29 0.73 0.04 0.49 0.51 1.09 0.23 sapiens, dickkopf (Xenopus laevis)homolog 1 (DKK1), mRNA./ PROD = dickkopf (Xenopus laevis) homolog 1/ FL= gb: AF177394.1 gb: NM_012242.1 gb: AF127563.1 AL096776 Human DNA —1.94 0.07 3.41 0.16 2.40 0.07 3.40 0.14 sequence from clone RP4-646B12on chromosome 1q42.11-42.3 Contains an FTH1 (ferritin, heavypolypeptide 1) (FTHL6) pseudogene, the gene for a novel Ras familyprotein, ESTs, STSs, GSSs and a putative CpG island/ FL = gb: AF282258.1b: NM_0212 BC005997 Homo — 1.12 0.25 0.95 0.44 2.04 0.07 1.51 0.22sapiens clone MGC: 14801, mRNA, complete cds./ PROD = Unknown (proteinfor MGC: 14801)/ FL = gb: BC005997.1 AF074979 Homo RGS20 −0.52 0.18−0.04 0.38 −1.30 0.09 −0.34 0.13 sapiens regulator of G proteinsignaling-Z (RGSZ1) mRNA, complete cds./ PROD = regulator of G proteinsignaling/ FL = gb: AF060877.2 gb: AF074979.1 gb: NM_003702.2 BF060767ESTs ADAMTS5 −0.36 0.15 0.80 0.33 0.22 0.14 0.40 0.56 AU151151 Homo LEPR1.72 0.18 2.38 0.80 1.85 0.10 1.71 0.69 sapiens cDNA FLJ13536 fis, clonePLACE1006521 L27624 Homo TFPI2 3.77 0.06 3.57 0.77 2.59 0.11 2.44 0.89sapiens tissue factor pathway inhibitor-2 mRNA, complete ds./ PROD =tissue factor pathway inhibitor-2/ FL = gb: D29992.1 gb: L27624.1 gb:NM_006528.1 gb: BC005330.1 NM_003174 Homo SVIL 3.15 0.09 3.68 0.44 2.910.01 3.27 0.46 sapiens supervillin (SVIL), transcript variant 1, mRNA./PROD = supervillin, isoform 1/ FL = gb: AF051851.1 gb: NM_003174.2 gb:AF051850.1 AF052127 Homo RELN −3.13 1.04 −1.36 0.26 −0.88 0.17 −0.400.11 sapiens clone 23850 mRNA sequence. AL031290 Human DNA — 0.38 0.061.39 0.46 0.40 0.05 0.95 0.44 sequence from clone 774124 on chromosome1q24.1-24.3 Contains protein similar to pregnancy- associated plasmaprotein A precursor neuronal igration protein ESTs, STS astrotactin,ESTs, STS and GSS AI129628 ESTs SAMD3 −0.30 0.18 −0.01 0.25 0.72 0.060.17 0.21 NM_016206 Homo VGLL3 −0.39 0.36 0.05 1.03 −0.31 0.29 −1.051.01 sapiens colon carcinoma related protein (LOC51159), mRNA./ PROD =colon carcinoma related protein/ FL = gb: NM_016206.1 gb: AF099505.1BE348291 ESTs — 1.62 0.06 −0.19 0.98 −3.65 0.10 −2.75 1.84 AW242720 HomoLOC143381 −1.62 0.34 −1.20 0.94 −0.08 0.24 −0.08 0.77 sapiens cDNAFLJ10561 fis, clone NT2RP2002672 NM_001146 Homo ANGPT1 1.86 0.06 2.090.81 1.10 0.16 0.87 1.05 sapiens angiopoietin 1 (ANGPT1), mRNA./ PROD =angiopoietin 1/ FL = gb: NM_001146.1 gb: D13628.1 gb: U83508.1 AU152579Homo PCSK5 2.41 0.14 2.47 0.86 2.04 0.15 1.11 1.20 sapiens cDNA FLJ13034fis, clone NT2RP3001232 M_006200 Homo PCSK5 3.01 0.21 2.88 0.92 2.280.11 1.52 1.25 sapiens proprotein convertase subtilisinkexin type 5(PCSK5), mRNA./ PROD = proprotein convertase subtilisinkexin type 5/FL =gb: U56387.2 gb: NM_006200.1 BF342661 KIAA0036 MAP2 −0.77 0.30 −0.190.44 0.12 0.17 −2.21 1.64 gene product AF063824 Homo TRPC4 −3.03 0.390.05 0.49 1.28 0.17 0.21 0.92 sapiens trp- related protein 4 truncatedvariant delta mRNA, complete cds./ PROD = trp- related protein 4truncated variant delta/ FL = gb: AF063824.1 AA723810 cDNA for LY6K 0.070.20 −0.13 1.23 0.15 0.14 −0.38 1.27 differentially expressed CO16 gene/FL = gb: BC001291.1 N29877 interleukin 14 — 1.94 0.13 0.51 0.48 0.780.64 −1.57 0.65 NM_007287 Homo MME 2.59 0.03 1.81 0.40 1.89 0.21 2.140.30 sapiens membrane metallo- endopeptidae (neutral endopeptidase,enkephalinae, CALLA, CD10) (MME), transcript variant 1bis, mRNA./ PROD =membrane metallo- endopeptidase/ FL = gb: NM_007288.1 gb: NM_007287.1gb: J03779.1 AB050856 Homo B3GALNT1 1.39 0.05 0.20 0.22 −0.13 0.21 −0.100.17 sapiens beta3GalNA cT-1 mRNA for globoside synthase, complete cds,clone: type 2./ PROD = globoside synthase/ FL = gb: AB050856.1 AI827455Homo BCL6B 0.65 0.31 0.62 0.21 1.07 0.24 0.90 0.29 sapiens cDNA:FLJ21042 fis, clone CAE11204 AF017987 Homo SFRP1 4.15 0.22 4.37 1.246.02 0.06 5.74 1.01 sapiens secreted apoptosis related protein 2 (SARP2)mRNA, omplete cds./ PROD = secreted apoptosis related protein 2/ FL =gb: AF056087.1 gb: NM_003012.2 gb: AF017987.1 gb: AF001900.1 AL117451Homo C1orf108 −0.73 0.20 0.68 0.24 1.06 0.05 0.82 0.21 sapiens mRNA;cDNA DKFZp586E2317 (from clone DKFZp586E2317). AW268357 ESTs, HighlyUSP32 0.73 0.20 0.36 0.19 0.70 0.03 0.09 0.18 similar to AF155116 1NY-REN-60 antigen (H.sapiens) BE465243 ESTs ARFGEF1 0.50 0.23 0.16 0.321.03 0.08 0.47 0.06 AA993400 ESTs ADAL 0.89 0.22 0.48 0.28 0.83 0.100.29 0.43 AI970898 Cluster Incl. ACACB 0.69 0.15 0.51 0.53 1.46 0.11−0.87 0.83 AI970898: wr21c03.x1 Homo sapiens cDNA,3 end/clone = IMAGE-2488324/ clone_end = 3/ gb = AI970898/ gi = 5767724/ ug = Hs.234898/len = 382 G026457 ESTs, KIAA1909 0.60 0.07 0.23 0.59 0.98 0.03 −2.111.28 Weakly similar to ALU5_HUMAN ALU SUBFAMILY SC SEQUENCECONTAMINATION WARNING ENTRY (H.sapiens) BC041933 Homo UBE3C 0.83 0.230.11 0.51 0.55 0.05 0.41 0.92 sapiens, clone IMAGE: 5300703, mRNA.AI638611 KIAA1373 STAMBPL1 2.20 0.17 1.37 0.58 2.32 0.08 −0.39 1.24protein AI341686 ESTs, Highly MTRF1 1.36 0.14 0.45 0.48 1.43 0.09 −1.090.99 similar to RF1M_HUMAN MITOCHONDRIAL PEPTIDE CHAIN RELEASE FACTOR 1PRECURSOR (H.sapiens) NM_003182 Homo TAC1 1.89 0.20 1.65 0.13 2.46 0.091.48 0.26 sapiens tachykinin, precursor 1 (substance K, substance P,neurokinin 1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptideK, neuropeptide gamma) TAC1), transcript variant beta, mRNA./ PROD =tachykinin 2 precursor, isoform beta/ FL = gb: U3 M80634 Human FGFR23.63 0.06 2.25 0.39 2.79 0.19 2.07 0.84 keratinocyte growth factorreceptor mRNA, complete cds./PROD = keratinocyte growth factor receptor/FL = gb: M80634.1 gb: NM_022969.1 gb: M97193.1 AK021452 Homo ZNF521−0.20 0.16 −1.15 1.12 1.31 0.05 0.36 0.18 sapiens cDNA FLJ11390 fis,clone HEMBA1000561, weakly similar to ZINC FINGER PROTEIN 91. AA541622ESTs SYNPO2 −0.49 0.30 −0.90 0.06 0.35 0.09 −2.58 0.93 N50714 ESTs —−1.13 0.13 −1.34 0.52 0.10 0.12 −0.21 0.28 NM_002674 Homo PMCH 0.62 0.241.10 0.48 1.64 0.03 0.09 0.80 sapiens pro- melanin- concentratinghormone (PMCH) mRNA./ PROD = pro- melanin- oncentrating hormone/ FL =gb: NM_002674.1 gb: M57703.1 BM666010 Homo LOC200169 1.36 0.16 0.45 0.741.49 0.12 −1.65 1.41 sapiens cDNA FLJ23803 fis, clone HEP22811. AI343467Homo — −0.44 0.08 −1.50 0.82 1.11 0.19 −0.64 0.20 sapiens cDNA FLJ11041fis, clone PLACE1004405 AA046424 ESTs, ACOT4 0.58 0.27 −0.94 0.66 −1.080.38 −1.09 0.25 Weakly similar to YZ28_HUMAN HYPOTHETICAL PROTEIN ZAP128(H.sapiens) R38389 olfactomedin OLFM1 4.27 0.11 3.02 0.76 4.53 0.07 2.170.96 related ER localized protein BF724270 ESTs — 2.03 0.22 0.72 0.422.36 0.06 0.01 0.97 NM_001819 Homo CHGB 1.40 0.18 0.50 0.75 1.66 0.05−0.74 1.03 sapiens chromogranin B (secretogranin 1) (CHGB), mRNA./ PROD= chromogranin B precursor/ FL = gb: BC000375.1 gb: NM_001819.1 AK026387Homo — 0.03 0.28 −0.05 0.52 1.23 0.26 0.52 0.67 apien CDNA: FLJ22734fis, clone HUV00109. BF062139 polymerase — 5.23 0.06 4.31 0.74 5.80 0.073.37 0.86 (RNA) III (DNA directed) (32kD)/ FL = gb: NM_006467.1 gb:U93868.1 BG540454 ESTs SCGB3A2 4.64 0.02 2.91 0.84 4.13 0.07 1.72 0.83AI659533 ArgAbl- SORBS2 0.02 0.12 1.12 0.71 2.17 0.05 −1.08 1.48interacting protein ArgBP2 AA531287 ESTs — 2.94 0.27 1.32 0.72 2.57 0.040.22 0.90 NM_013243 Homo SCG3 3.30 0.20 2.13 0.95 3.67 0.19 0.87 1.06sapiens secretogranin III (SCG3), mRNA./ PROD = secretogranin III/ FL =gb: AF078851.1 gb: NM_013243.1 AI307586 Homo C10orf95 0.34 0.22 −1.150.24 −0.49 0.16 −3.61 0.99 sapiens mRNA; CDNA DKFZp566H0124 (from cloneDKFZp566H0124) BC032004 Homo GRIA3 −0.02 0.23 −0.15 0.33 0.47 0.04 0.170.07 sapiens, Similar to glutamate receptor, ionotrophic, AMPA 3, cloneIMAGE: 4753 74, mRNA. AW205739 ESTs, TYW3 0.47 0.01 0.81 1.35 3.72 0.182. 6 1.15 Weakly similar to ORF YGL050w (S.cerevisiae) NM_153262 HomoSYT14 −0.01 0.17 −0.84 0.51 0.00 0.18 −3.88 1.64 sapiens hypotheticalprotein FLJ34198 (FLJ34198), mRNA./ FL = gb: NM_153262.1 AA156873albumin PELO 0.77 0.12 −0.23 0.93 0.65 0.14 −2.33 1.01 BC012375 HomoARSG −0.46 0.48 −0.78 0.42 0.57 0.22 −0.41 0.55 sapiens, Similar toKIAA1001 protein, clone MGC: 8996 IMAGE: 3882163, mRNA, completecds./PROD = Similar to KIAA1001 protein/ FL = gb: AB023218.1 gb:NM_014960.1 gb: BC012375.1 AA843242 ESTs BNC2 2.12 0.37 1.03 0.47 2.890.06 0.43 0.80 BF792954 ESTs HDLBP −1.53 1.04 −2.75 0.91 0.30 0.16 −0.720.26 AA780067 heparan HS3ST3B1 0.41 0.19 0.30 0.61 2.61 0.18 0.78 0.60sulfate (glucosamine) 3-O-sulfotransferase 3B1 AA909330 ESTs RP1-32F7.20.98 0.17 −1.67 0.51 0.79 0.02 −0.38 0.12 F141339 Homo ZNF521 −0.41 0.12−0.97 0.93 1.67 0.11 −0.31 0.80 sapiens LYST- interacting protein LIP3mRNA, partial cds./ PROD = LYST- interacting protein LIP3 BF435123bromodomain — 2.28 0.17 1.31 0.51 2.70 0.12 1.30 1.00 and PHD fingercontaining, 3 AK056212 Homo — 1.03 0.14 0.14 0.27 1.10 0.07 −1.41 0.51sapiens CDNA FLJ31650 fis, clone NT2RI2004079. NM_001446 Homo FABP7 2.070.15 0.74 0.29 2.20 0.04 1.08 0.16 sapiens fatty acid binding protein 7,brain (FABP7), mRNA./ PROD = fatty acid binding protein 7, brain/ FL =gb: U81235.1 gb: D88648.1 gb: U51338.1 gb: NM_001446.1 gb: D50373.1AW051591 ESTs, RNF175 1.41 0.32 0.22 0.10 2.60 0.06 0.96 0.20 Moderatelysimilar to unnamed protein product (H.sapiens) BC041970 Homo C9orf1220.74 0.03 −0.35 0.49 0.76 0.06 −2.31 1.25 sapiens, lone IMAGE: 5302687,mRNA. BC013077 Homo — 2.51 0.11 0.80 1.15 2.85 0.06 0.08 0.89 sapiens,clone IMAGE: 3459334, mRNA. AW572379 ESTs — 1.80 0.03 0.60 0.84 1.450.07 1.88 0.53 BE644917 nuclear XIST −3.88 1.26 −0.54 1.28 1.94 0.13−0.20 1.94 receptor subfamily 1, group I, member 3 NM_171999 Homo SALL32.97 0.18 2.04 0.71 3.79 0.08 1.02 0.74 sapiens sal- like 3 (Drosophila)(SALL3), mRNA./ PROD = sal-like 3/ FL = gb: NM_171999.1 AI654224 ESTs —0.46 0.36 0.14 0.44 1.03 0.07 −0.81 0.85 AA167449 nuclear XIST −3.080.10 −0.47 2.59 4.59 0.07 2.80 1.86 receptor subfamily 1, group I,member 3 BF977837 KIAA0527 SUSD5 1.64 0.02 0.50 0.68 1.96 0.16 −1.521.66 protein BC029425 Homo FILIP1 −0.83 0.45 −0.03 0.62 0.76 0.20 −0.290.44 sapiens, Similar to KIAA1275 protein, clone IMAGE: 4616553, mRNA.AI978754 ESTs — 2.76 0.14 1.54 0.57 3.57 0.06 2.31 0.69 AA628440 nuclearXIST 0.18 0.13 1.42 1.52 4.80 0.02 3.01 1.50 receptor subfamily 1, groupI, member 3 L36861 L36861/ — 2.75 0.17 2.01 0.82 3.63 0.09 3.00 0.97FEATURE = expanded_cds/ DEFINITION = HUMGCAPB Homo sapiens guanylatecyclase activating protein (GCAP) gene exons 1-4, complete cds AW023227ESTs MKX −0.91 0.60 −1.31 0.06 0.27 0.13 −2.90 0.52 NM_021614 Homo KCNN23.45 0.13 2.52 0.83 4.40 0.14 1.54 0.80 sapiens potassium intermediatesmall conductance calcium- activated channel, subfamily N, member 2(KCNN2), mRNA./ PROD = potassium intermediate small conductance calcium-activated channel, subfamily N, member 2/ FL = gb: NM_021614.1 gb:AF239613.1 NM_000956 Homo PTGER2 −0.71 0.71 −0.73 0.57 0.63 0.33 −2.380.61 sapiens prostaglandin E receptor 2 (subtype EP2), 53kD PTGER2),mRNA./ PROD = prostaglandin E receptor 2 (subtype EP2), 53kD/ FL = gb:U19487.1 gb: NM_000956.1 NM_013381 Homo TRHDE 1.96 0.16 0.25 0.36 1.810.03 0.82 0.23 sapiens thyrotropin- releasing hormone degradingectoenzyme (TRHDE), mRNA./ PROD = thyrotropin- releasing hormonedegrading- ectoenzyme/ FL = gb: AF126372.1 gb: NM_013381.1 NM_016354Homo SLCO4A1 2.18 0.06 1.01 0.37 2.35 0.05 −0.91 1.23 sapiens solutecarrier family 21 (organic anion transporter), member 12 (SLC21A12),mRNA./ PROD = organic anion transporter OATP-E/ FL = gb: AB031051.1 gb:NM_016354.1 b: AF205072.1 gb: AF187817.1 BC028359 Homo sapiens, ZNF141−1.05 0.39 −0.59 0.65 1.25 0.17 −1.48 0.72 clone IMAGE: 4828836, mRNA.AI193252 ESTs, LRRN6A 4.38 0.11 2.47 0.43 4.23 0.11 1.90 0.37 Weaklysimilar to AF133270 1 SLIT2 (H.sapiens) H09780 Human — 2.04 0.19 0.560.07 2.50 0.11 0.12 0.40 (clone CTG- A4) mRNA sequence BC040605 Homo —2.64 0.16 1.61 1.08 3.59 0.09 0.45 1.14 sapiens, clone IMAGE: 5271039,mRNA. AW057589 ESTs — −0.96 0.26 −2.77 0.81 0.25 0.24 −1.21 0.35 M31213Human RET 0.42 0.10 −1.86 0.83 1.77 0.12 −0.64 0.15 papillary thyroidcarcinoma- encoded protein mRNA, complete cds./FL = gb: M31213.1 Z92546Human DNA — 0.58 0.13 −1.16 0.54 1.07 0.12 −1.27 0.14 sequence fromclone CTA-65A6 on chromosome 22q11-12 Contains the 3 part of the genefor the ortholog of rat CAIN KIAA0330), the gene for a novel Sushidomain (SCR repeat) containing protein similar to Mucins, ESTs, an STS,GSSs and two . . . AA974416 protein PPP2R2B 4.02 0.16 3.52 0.23 5.220.07 3.12 0.15 phosphatase 2 (formerly 2A), regulatory subunit B (PR52), beta isoform AW138143 ESTs SORBS2 3.44 0.16 2.89 0.88 4.47 0.101.88 1.15 NM_014862 Homo ARNT2 1.29 0.08 −0.68 0.97 2.00 0.08 0.02 0.24sapiens KIAA0307 gene product (KIAA0307), mRNA./ PROD = KIAA0307 geneproduct/ FL = gb: AB002305.1 gb: NM_014862.1 AI765540 ESTs — 1.28 0.220.07 0.39 1.50 0.07 −0.11 0.26 NM_018013 Homo FLJ10159 1.30 0.21 0.380.39 2.89 0.02 0.62 0.21 sapiens hypothetical protein FLJ10159(FLJ10159), mRNA./PROD = hypothetical protein FLJ10159/ FL = gb:NM_18013.1 BF382322 ESTs, — −0.40 .09 −2.09 .67 −0.12 .08 −1.73 .50Weakly similar to unnamed protein product (H.sapiens) AV699347 nuclearXIST −1.37 0.38 0.45 1.69 4.28 .02 2.25 1.53 receptor subfamily 1, groupI, member 3 BC011549 Homo ATP5S 0.69 0.19 0.96 0.68 2.57 .29 −0.02 0.52sapiens, clone MGC: 19945 IMAGE: 4554461, mRNA, complete cds./ PROD =Unknown (protein for MGC: 19945)/ FL = gb: BC011549.1 NM_001889 HomoCRYZ −1.68 0.26 0.23 1.38 3.75 .07 1.78 1.11 sapiens crystallin, zeta(quinone reductase) (CRYZ), mRNA./ PROD = crystallin, zeta (quinonereductase)/ FL = gb: NM_001889.1 gb: L13278.1 gb: S58039.1 BC002665 HomoPLP1 4.57 0.15 2.95 0.64 4.70 .06 2.32 0.52 sapiens, proteolipid protein(Pelizaeus-Merzbacher disease, spastic paraplegia 2, uncomplicated),clone MGC: 3940, mRNA, complete cds./ PROD = proteolipid protein(Pelizaeus- Merzbacherd isease, spastic paraplegia 2, uncomplicated), FL= gb: BC002665.1 NM_001243 Homo TNFRSF8 3.75 0.03 1.45 0.35 3.24 0.110.89 0.46 sapiens tumor necrosis factor receptor superfamily, member 8(TNFRSF8), mRNA./ PROD = CD30 antigen (Ki-1 antigen)/ FL = gb:NM_001243.1 gb: D86042.1 gb: M83554.1 NM_001195 Homo BFSP1 0.57 0.33−1.65 1.30 0.60 0.14 −0.86 0.73 sapiens beaded filament structuralprotein 1, filensin (BFSP1), RNA./ PROD = filensin/ FL = gb: AF039655.1gb: NM_001195.2 gb: Y16717.2 NM_024582 Homo FAT4 2.16 0.27 −0.49 0.301.54 0.28 −1.35 1.01 sapiens hypothetical protein FLJ23056 (FLJ23056),mRNA./ PROD = hypothetical protein FLJ23056/ FL = gb: NM024582.1NM_000767 Homo CYP2B6 2.60 0.18 0.87 0.96 1.47 0.12 −0.69 1.03 sapienscytochrome P450, subfamily IIB (phenobarbital- inducible), polypeptide 6(CYP2B6), mRNA./ PROD = cytochrome P450, subfamily IIB(phenobarbital-inducible), polypeptide 6/ FL = gb: NM_000767.2 gb: AF182277.1 gb:M29874.1 AW665509 ESTs MGC42174 −0.10 0.33 −0.87 0.72 1.20 0.08 −2.601.40 NM_001104 Homo sapiens ACTN3 2.12 0.13 1.64 0.28 3.35 0.03 1.670.36 ctinin, alph 3 (ACTN3), mRNA./ PROD = skeletal muscle specificactinin, alpha 3/ FL = gb: M86407.1 gb: NM_001104.1 NM_021069 HomoSORBS2 −0.49 0.43 0.30 1.36 2.03 0.07 −1.13 1.15 sapiens ArgAbl-interacting protein ArgBP2 (ARGBP2), transcript variant 2, mRNA./ PROD =ArgAbl- interacting protein 2, isoform 2/ FL = gb: AB018320.1 gb:NM_021069.1 T65020 ESTs — 0.51 0.16 −0.84 0.26 1.10 0.15 −1.50 0.38NM_152647 Homo GALK2 0.08 0.56 −1.42 0.52 0.03 0.17 −2.34 0.91 sapienshypothetical protein FLJ32800 (FLJ32800), mRNA./ FL = gb: NM_152647.1BC036917 Homo C6orf141 −0.82 0.30 −1.18 0.40 0.88 0.11 −2.18 0.62sapiens, clone MGC: 46457 IMAGE: 5201433, mRNA, complete ds./PROD =Unknown MGC: 46457) (protein for MGC: 46457)/ FL = 6gb: BC03917.1AI969112 Homo PHIP −1.07 0.13 −2.88 0.91 −0.37 0.02 −2.23 0.33 sapiens,clone IMAGE: 5260603, mRNA, partial cds AW449813 KIAA0918 SLITRK5 0.320.26 −1.89 0.50 0.46 0.10 −0.57 0.13 protein AW044658 ESTs — 0.16 0.16−1.47 0.25 0.90 0.14 −0.89 0.16 AI694300 ESTs — −0.52 0.02 −1.34 0.520.27 0.08 −1.00 0.08 NM_017631 Homo FLJ20035 1.35 0.13 0.05 0.19 1.510.17 0.32 0.15 sapiens hypothetical protein FLJ20035 (FLJ20035), mRNA./PROD = hypothetical protein FLJ20035/ FL = gb: NM_017631.1 AF298547 HomoNALP2 1.54 0.08 1.22 1.61 5.04 0.05 3.23 1.36 sapiens nucleotide-binding site protein 1 mRNA, complete cds./ PROD = nucleotide- bindingsite protein 1/ FL = gb: AF298547.1 NM_030631 Homo SLC25A21 1.96 0.250.33 0.81 2.47 0.11 −0.28 0.63 sapiens oxodicarboxylate carrier ODC1),mRNA./ PROD = oxodicarboxylate carrier/ FL = gb: NM_030631.1 BF196255ESTs — 2.94 0.05 1.18 0.43 3.15 0.08 0.54 0.57 NM_003247 Homo THBS2 2.620.20 1.04 0.28 2.70 0.04 0.27 0.45 sapiens thrombospondin 2 (THBS2),mRNA./ PROD = thrombospondin 2/ FL = gb: NM_003247.1 gb: L12350.1NM_001446 Homo FABP7 0.95 0.09 −0.75 0.37 0.20 0.22 −1.73 0.99 sapiensfatty acid binding protein 7, brain (FABP7), mRNA./ PROD = fatty acidbinding protein 7, brain/ FL = gb: U81235.1 gb: D88648.1 gb: U51338.1gb: NM_001446.1 gb: D50373.1 AW004016 ESTs ST6GAL2 1.57 0.14 0.89 0.323.30 0.07 1.48 0.43 AW072790 contactin 1 CNTN1 0.50 0.20 0.18 0.98 1.840.10 1.01 1.39 AL512686 Homo GNAO1 2.16 0.50 −0.28 1.03 1.67 0.18 −1.120.37 sapiens mRNA; cDNA DKFZp7611177 (from clone DKFZp7611177). AI638063ESTs CBX5 0.49 0.16 −0.34 0.34 0.98 0.11 −2. 1 1.22 AU157049 HomoLOC153346 2.24 0.20 0.34 0.47 2.24 0.09 −0.44 0.96 sapiens cDNA FLJ14284fis, clone PLACE1005898 NM_002738 Homo PRKCB1 0.99 0.28 −0.15 0.45 1.420.08 −1.02 1.04 sapiens protein kinase C, beta 1 (PRKCB1), mRNA./ PROD =protein kinase C, beta 1/ FL = gb: NM_002738.1 AI185136 ESTs DYDC2 0.120.31 −0.80 0.08 0.72 0.08 −0.60 0.09 AI928037 ESTs RPIB9 −0.55 0.55−2.71 0.74 0.49 0.12 −0.89 0.27 NM_016179 Homo sapiens TRPC4 −1.51 0.85−1.87 1.42 1.71 0.11 −0.13 1.23 transient receptor potential channel 4(TRPC4), mRNA./ PROD = transient receptor potential 4/ FL = gb:NM_016179.1 gb: AF175406.1 AW138143 ESTs SORBS2 3.29 0.13 2.35 1.05 4.280.10 1.32 1.07 NM_001876 Homo sapiens CPT1A 1.63 0.18 −0.15 0.21 1.320.07 −0.20 0.33 carnitine palmitoyltran- sferase I, liver (CPT1A),nuclear gene encoding mitochondrial protein, mRNA./ PROD = livercarnitine palmitoyltran- sferase I/ FL = gb: L39211.1 gb: NM_001876.1AA903862 ESTs C20orf54 1.61 0.15 −1.17 0.30 0.68 0.21 −1.49 0.23AI670947 phosphatidylinositol- — 3.91 0.05 0.55 1.54 3.33 0.10 1.17 0.684-phosphate 5- kinase, type I, beta AV648405 polymerase — −0.01 0.110.23 0.52 1.73 0.14 −1.23 1.15 (RNA) III (DNA directed) (32kD) BF435123bromodomain — 1.80 0.13 −1.35 1.01 1.14 0.03 0.37 0.24 and PHD fingercontaining, 3 NM_016582 Homo SLC15A3 1.77 0.16 −0.88 1.35 1.30 0.23−2.04 1.23 sapiens peptide transporter 3 (LOC51296), mRNA./ PROD =peptide transporter 3/ FL = gb: NM_016582.1 gb: AB020598.1 AI830490glycerol GK 0.61 0.01 −0.65 0.79 1.25 0.20 −0.38 0.33 kinase AW134979HSPC156 STXBP6 2.28 0.03 0.55 0.28 2.05 0.03 0.30 0.44 protein AI354636ESTs — 2.81 0.11 0.68 1.17 3.02 0.20 0.19 0.76 BE672659 ESTs — 0.20 0.64−0.43 0.61 1.98 0.12 0.69 0.45 AF284095 Homo ADRA2A 0.63 0.21 −0.74 0.981.85 0.03 0.55 0.31 sapiens Iph-2A adrenergic receptor mRNA, completecds./ PROD = alpha-2A adrenergic receptor/ FL = gb: AF284095.1 gb:NM_000681.1 NM_021136 Homo RTN1 −0.30 0.03 −1.39 0.35 0.80 0.07 −1.230.39 sapiens reticulon 1 (RTN1), mRNA./ PROD = reticulon 1/ FL = gb:L10333.1 gb: L10334.1 gb: NM_021136.1 NM_014729 Homo TOX 0.34 0.03 −0.460.12 0.90 0.18 −1.86 0.40 sapiens KIAA0808 gene product (KIAA0808),mRNA./ PROD = KIAA0808 gene product/ FL = gb: AB018351.1 gb: NM_014729.1BE674118 ESTs — 0.78 0.07 −1.32 0.73 0.28 0.19 −0.96 0.74 BC026969 HomoWDR67 2.95 0.11 0.75 0.92 3.08 0.08 0.45 0.61 sapiens, clone IMAGE:5116073, mRNA, partial cds. AF131783 Homo PAP2D −0.42 0.26 −2.00 1.050.31 0.22 −1.56 0.70 sapiens lone 25181 mRNA sequence. U11058 HomoKCNMA1 1.56 0.09 −0.16 1.08 2.29 0.18 −0.79 0.86 sapiens largeconductance calcium- and voltage- dependent potassium channel alphasubunit (MaxiK) mRNA, complete cds./ PROD = large conductance calcium-and voltage- dependent- potassium channel alpha subunit/ FL = gb:U23767.1 gb: NM_002247.1 gb: AF025999 BF510715 fibroblast — 2.67 0.270.84 0.17 3.29 0.01 0.64 0.51 growth factor 4 (heparin secretorytransforming protein 1, Kaposi sarcoma oncogene)/ FL = gb: M17446.1 gb:NM_002007.1 BE897866 ESTs ACADSB 1.41 0.23 0.08 0.34 3.10 0.10 −0.270.88 I735586 ESTs LOC152573 0.60 0.13 0.00 1.14 2.84 0.07 1.12 1.30AL573058 complement C1R 0.92 0.14 −1.67 0.38 0.73 0.02 −0.96 0.77component 1, r subcomponent AF429305 Homo RMST −0.48 0.03 −2.49 0.51−0.25 0.20 −1.89 0.27 sapiens C23up NCRMS mRNA, partial sequence;alternatively spliced. BF195118 ESTs, ATP5J 0.09 0.13 −0.92 0.42 1.570.16 −0.53 0.08 Weakly similar to ALU7_HUMAN ALU SUBFAMILY SQ SEQUENCECONTAMINATION WARNING ENTRY (H.sapiens) NM_005460 Homo SNCAIP 1.76 0.16−0.84 0.52 1.61 0.19 −0.92 0.88 sapiens synuclein, alpha interactingprotein (synphilin) (SNCAIP), mRNA./ PROD = synuclein alpha interactingprotein/ FL = gb: AF076929.1 gb: NM_005460.1 NM_024893 Homo C20orf391.16 0.29 −0.56 0.42 2.36 0.15 −0.06 0.17 sapiens hypothetical roteinFLJ14220 (FLJ14220), mRNA./ PROD = hypothetical protein FLJ14220/ FL =gb: NM024893.1 NM_022034 Homo CUZD1 1.31 0.08 0.80 0.28 2.78 0.07 0.240.58 sapiens estrogen regulated gene 1 (ERG-1), mRNA./ PROD = estrogenregulated gene 1/ FL = gb: AF305835.1 gb: NM_022034.1 NM_014788 HomoTRIM14 3.12 0.09 1.22 0.59 3.54 0.08 1.02 0.45 sapiens KIAA0129 geneproduct (KIAA0129), mRNA./ PROD = KIAA0129 gene product/ FL = gb:D50919.1 gb: NM_014788.1 AV646597 ESTs, XIST −0.77 0.18 0.06 1.55 3.780.25 2.28 0.98 Weakly similar to ALU7_HUMAN ALU SUBFAMILY SQ SEQUENCECONTAMINATION ENTRY ARNING ENTRY (H.sapiens) BF062629 DKFZP586E1621TMEM158 4.00 0.19 2.41 0.27 4.87 0.00 1.93 0.19 protein AW440492 ATPase,ATP1A2 1.33 0.07 −2.12 1.07 1.27 0.06 −0.53 0.11 Na + K + transporting,alpha 2 (+) polypeptide/ FL = gb: NM_000702.1 AF283777 Homo CD72 −0.080.45 −1.88 1.19 0.41 0.24 −1.42 1.13 sapiens clone TCBAP0702 mRNAsequence. NM_005375 Homo MYB 2.11 0.17 −0.52 0.18 2.24 0.03 0.10 0.08sapiens v- myb avian myeloblastosis viral oncogene homolog (MYB), mRNA./PROD = v- myb avian myeloblastosis viral oncogenehomolog/ FL = gb:NM_005375.1 gb: AF104863.1 gb: M15024.1 NM_017671 Homo C20orf42 3.190.12 0.97 0.70 3.26 0.07 0.16 0.94 sapiens hypothetical protein FLJ20116(FLJ20116), mRNA./ PROD = hypothetical protein LJ20116/ FL = gb:NM_017671.1 BG283790 ESTs MATR3 2.27 0.11 0.95 0.57 3.53 0.04 0.89 0.51NM_000702 Homo ATP1A2 2.12 0.17 0.76 0.38 2.93 0.16 0.51 0.12 sapiensATPase, Na + K + transporting, alpha 2(+) polypeptide (ATP1A2), mRNA./PROD = ATPase, Na + K + transporting, alpha 2 (+)polypeptide/ FL = gb:NM_000702.1 NM_025135 Homo FHOD3 1.02 0.18 0.80 0.40 2.34 0.10 −0.770.72 sapiens hypothetical protein FLJ22297 (KIAA1695), mRNA./ PROD =hypothetical protein KIAA1965/ FL = gb: NM_025135.1 NM_012281 Homo KCND21.67 0.30 0.05 0.15 2.70 0.09 −0.26 0.38 sapiens potassium voltage-gatedchannel,/shal- related subfamily, member 2 (KCND2), mRNA./ PROD =potassium voltage-ated channel, Shal- relatedsubfamily, member 2/ FL =gb: NM_012281.1 gb: AB028967.1 gb: AF121104.1 BF449063 collagen, COL14A11.61 0.15 0.30 0.38 2.68 0.17 −0.33 0.68 type XIV, alpha 1 (undulin)AA280904 ESTs C9orf39 0.23 0.21 −1.58 1.26 1.13 0.14 −0.78 0.42NM_022467 Homo CHST8 2.11 0.20 −0.51 0.30 2.17 0.10 0.48 0.02 sapiens N-acetylgalacto- samine-4-O- sulfotransferase (GALNAC-4- ST1), mRNA./ PROD= N- acetylgalacto- samine-4-O- sulfotransferase/ FL = gb: NM_022467.1gb: AF300612.1 BE468066 ESTs RMST 3.26 0.09 1.49 0.10 3.80 0.09 1.630.08 AL120674 ESTs — 1.00 0.11 −1.35 1.73 1.55 0.15 −2.20 0.86 NM_133329Homo KCNG3 2.18 0.27 0.74 0.29 3.67 0.01 0.26 0.28 sapiens potassiumvoltage- gated channel, subfamily G, member 3 (KCNG3) transcript variant1, RNA./PROD = potassium voltage- gated channel, subfamily G, member 3isoform 1/ FL = gb: AF454548.1 gb: AF348982.1 gb: AB070604.1 gb:NM_133328.4 AI742043 ESTs — 0.94 0.34 −0.76 0.37 1.77 0.10 −1.45 0.52NM_005103 Homo FEZ1 2.80 0.11 2.08 0.25 4.98 0.06 2.20 0.26 sapiensfasciculation and elongation protein zeta 1 (zygin I) (FEZ1), transcriptvariant 1, mRNA./ PROD = zygin 1, isoform 1/ FL = gb: U60060.1 gb:U69139.1 gb: NM_005103.2 NM_000277 Homo PAH 0.65 0.11 −1.08 0.08 1.460.18 −0.42 0.12 sapiens phenylalanine hydroxylase (PAH), mRNA./ PROD =phenylalanine hydroxylase/ FL = gb: U49897.1 gb: NM_000277.1 BF698797ESTs — 0.93 0.10 −0.74 0.11 2.07 0.10 −0.40 0.44 BF437747 ESTs,C20orf118 4.26 0.07 1.99 0.45 4.57 0.09 1.75 0.39 Weakly similar toALU7_HUMAN ALU SUBFAMILY SQ SEQUENCE CONTAMINATION WARNING ENTRY(H.sapiens) NM_003020 Homo sapiens SCG5 4.03 0.14 2.53 0.32 5.01 0.123.11 0.24 secretory granule, neuroendocrine protein 1 (7B2 protein)(SGNE1), mRNA./ PROD = secretory granule, neuroendocrine protein 1(7B2protein)/ FL = gb: BC0053489.1 gb: NM_003020.1 NM_002800 Homo PSMB91.38 0.10 0.27 0.34 2.05 0.07 −1.43 1.14 sapiens proteasome (prosome,macropain) subunit, beta type, 9 (large multi- functional protease 2)(PSMB9), mRNA./ PROD = proteasome (prosome, macropain) subunit,betatype, 9 (large multifunctional protease 2)/FL = gb: U01025.1 gb:NM_002800.1 BE972639 ESTs LOC646326 0.00 0.15 −2.71 0.55 0.33 0.22 −2.240.61 BC044830 Homo C10orf96 1.38 0.12 −0.32 0.82 2.28 0.06 −1.39 0.90sapiens, Similar to RIKEN cDNA 1700011F14 gene, clone MGC: 35062 IMAGE:5166167, mRNA, complete cds./PROD = Similar to RIKEN CDNA 1700011F14gene/FL = gb: BC044830.1 AI961231 KIAA0808 TOX 3.28 0.13 1.23 0.06 4.000.01 1.13 0.37 gene product/ FL = gb: AB018351.1 gb: NM_014729.1 U17496Human PSMB8 3.43 0.21 0.46 1.17 3.30 0.06 0.09 0.64 proteasome subunitLMP7 (allele LMP7B) mRNA, complete cds./ PROD = proteasome subunit LMP7/FL = gb: U17497.1 gb: U17496.1 N23651 ESTs SDK2 −1.16 0.31 −2.65 0.421.27 0.09 −2.07 0.75 NM_007015 Homo LECT1 4.83 0.12 2.27 0.84 5.36 0.091.83 0.69 sapiens chondromodulin I precursor (CHM-I), mRNA./ PROD =chondromodulin I precursor/ FL = gb: NM_007015.1 gb: AB006000.1NM_015474 Homo SAMHD1 2.63 0.17 0.57 0.33 2.89 0.09 0.29 0.22 sapiensDKFZP564A032 protein (DKFZP564A032), mRNA./PROD = DKFZP564A032 protein/FL = gb: AF228421.1 gb: AL050267.1 gb: AB013847.1 gb: NM_015474.1AF147427 Homo SAMHD1 1.33 0.03 −0.99 0.88 1.35 0.08 −2.30 0.97 sapiensfull length insert cDNA clone YP80A10. NM_004688 Homo NMI 3.54 0.04 0.960.07 3.80 0.07 1.13 0.34 sapiens N- myc (and STAT) interactor (NMI),mRNA./ PROD = N- myc and STAT interactor/ FL = gb: BC001268.1 gb:NM_004688.1 gb: U32849.1 AB040812 Homo PAK7 −1.37 0.60 −3.89 1.62 −0.490.07 −4.18 0.12 sapiens mRNA for protein kinase PAK5, complete cds./PROD = protein kinase PAK5/ FL = gb: AB040812.1 AI985987 ESTs, SCNN1G−0.28 0.31 −0.99 0.35 0.12 0.19 −2.70 0.59 Moderately similar toALU1_HUMAN ALU SUBFAMILY J SEQUENCE CONTAMINATION WARNING ENTRY(H.sapiens) NM_001877 Homo CR2 1.50 0.13 −0.43 1.05 2.35 0.14 −1.44 1.51sapiens complement component (3dEpstein Barr virus) receptor 2 (CR2),mRNA./ PROD = comlement component (3dEpstein Barr virus)receptor 2/ FL =gb: NM_001877.1 gb: M26004.1 NM_001351 Homo DAZL 1.69 0.24 −0.81 0.761.93 0.12 −0.73 0.06 sapiens deleted in azoospermia- like (DAZL), mRNA./PROD = deleted in azoospermia-like/ FL = gb: U66726.2 gb: NM_001351.1gb: U65918.1 gb: U66078.1 NM_022168 Homo IFIH1 −2.03 1.08 −2.76 0.83−0.10 0.15 −2.15 0.40 sapiens melanoma differentiation associatedprotein-5 (MDA5), mRNA./ PROD = melanoma differentiation associatedprotein-5/ FL = gb: AY017378.1 gb: NM_022168.1 gb: AF095844.1 AF052108Homo LOC157627 1.49 0.33 0.22 1.02 2.26 0.40 −1.33 1.19 sapiens clone23687 mRNA equence. AI056877 Human DNA LOC200230 0.11 0.30 −2.37 0.941.15 0.18 −1.35 0.75 sequence from clone RP4-530115 on chromosome 20.Contains the 3 end of the PTPN1 gene for protein tyrosine phosphatase,non- receptor type 1 (EC 3.1.3.48), the gene for a novel protein similarto placental protein DIFF40, an RPL36 (60S Ribos NM_002522 Homo NPTX12.35 0.25 −0.48 0.19 2.17 0.09 −1.19 0.83 sapiens neuronal pentraxin I(NPTX1), mRNA./ PROD = neuronal pentraxin I precursor/ FL = gb:NM_002522.1 gb: U61849.1 N21096 ESTs STXBP6 2.42 0.16 0.99 0.08 3.690.09 1.14 0.04 AI693516 ESTs COL14A1 0.39 0.23 −1.63 0.96 0.96 0.17−1.81 0.67 NM_018043 Homo sapiens TMEM16A −0.90 0.37 −1.93 0.70 0.250.43 −3.93 0.49 hypothetical protein FLJ10261 (FLJ10261), mRNA. PROD =hypothetical protein FLJ10261/ FL = gb: NM_018043.1 AF110400 Homo FGF192.12 0.14 −0.08 0.34 2.18 0.04 −0.91 0.18 sapiens fibroblast growthfactor 19 (FGF19) mRNA, complete cds./ PROD = fibroblast growth factor19/ FL = gb: AF110400.1 gb: NM_005117.1 gb: AB018122.1 AK098525 HomoHHIP 1.76 0.14 −0.57 0.63 3.32 0.06 −0.11 0.15 sapiens cDNA FLJ25659fis, clone TST00427, highly similar to Mus musculus hedgehog-interacting protein (Hip) mRNA. AV715309 ESTs, C20orf118 4.28 0.13 1.890.39 4.76 0.10 1.48 0.42 Weakly similar to ALU7_HUMAN ALU SUBFAMILY SQSEQUENCE CONTAMINATION WARNING ENTRY H.sapiens) U96136 Homo sapiensCTNND2 0.78 0.11 −1.33 0.87 1.18 0.12 −1.17 0.67 delta-catenin mRNA,complete cds./PROD = delta-catenin/ FL = gb: NM_001332.1 gb: U72665.1gb: AB013805.1 gb: U96136.1 gb: AF035302.1 NM_002590 Homo PCDH8 −0.800.34 −0.83 0.82 2.18 0.02 0.12 0.33 sapiens protocadherin 8 (PCDH8),mRNA./PROD = protocadherin 8/ FL = gb: NM_002590.2 gb: AF061573.2AF107846 Homo sapiens — 0.44 0.20 −0.25 0.12 2.12 0.14 −2.56 0.81neuroendocrine- specific Golgi protein p55 (XLalphas) gene, exon XL2 andcomplete cds BG169832 adenylate AK5 0.62 0.36 −2.79 0.50 1.03 0.36 −1.940.32 kinase 5/FL = gb: NM_012093.1 gb: AF062595.1 BE968806 ESTs, ATP5S−1.15 0.14 −3.03 0.71 −0.34 0.15 −4.73 0.74 Weakly similar to LU4_HUMANALU SUBFAMILY SB2 SEQUENCE CONTAMINATION WARNING ENTRY (H.sapiens)BU729850 hypothetical JAKMIP2 2.30 0.07 0.43 1.78 3.86 0.04 −1.56 1.92protein LOC153469 AL832535 Homo LOC157627 2.25 0.18 −0.04 1.00 3.14 0.25−1.50 1.19 sapiens mRNA; cDNA DKFZp547J1816 (from clone DKFZp547J1816).NM_000439 Homo PCSK1 −0.63 0.18 −2.68 0.53 0.69 0.30 −3.19 1.03 sapiensproprotein convertase subtilisinkexin type 1 (PCSK1), mRNA./ PROD =proprotein convertase subtilisinkexin type 1/ FL = gb: NM_000439.2 gb:M90753.1 M34455 Human INDO 4.39 0.23 1.58 0.09 5.44 0.08 1.69 0.13interferon- gamma- inducible indoleamine 2,3- dioxygenase (IDO) mRNA,complete ds./ FL = gb: NM_002164.1 gb: M34455.1 AB014737 Homo SMOC2 1.250.12 −2.04 0.48 1.94 0.06 −2.27 1.12 sapiens mRNA for SMAP-2b, completecds./ PROD = SMA P-2b/ FL = gb: AB014737.1 BM682352 Homo HCN1 −0.41 0.14−4.38 1.19 0.35 0.31 −3.88 0.32 sapiens cDNA FLJ37204 fis, cloneBRALZ2006976.

TABLE V RELATIVE EXPRESSION OF DEFINITE ENDODERM MARKERS FOR CONDITIONSOUTLINED IN EXAMPLE 10. ALL VALUES WERE NORMALIZED TO GROUP 1 (CONTROL).Sox17 CXCR4 Goosecoid HNF-3B SOX-2 Oct-4 Group 1- 1 1 1 1 1 1 controlGroup 2 45 19.5 2.8 0.64 0.91 1.1 Group 3 45 30 2.9 0.74 0.70 0.76 Group4 8 14 2.7 1.11 0.18 0.36 Group 5 23 16 3.1 1.76 0.16 0.41 Group 6 415.8 3.0 1.87 0.61 0.57 Group 7 25 19.5 2.7 0.62 0.34 0.48 Group 8 6 15.92.9 2.0 0.13 0.43 Group 9 1 1.4 0.9 0.89 1.2 0.85 Group 10 22 1.5 1.41.20 1.36 0.68 Group 11 54 23 2.5 0.71 0.66 0.65 Group 12 68 0.7 0.91.51 0.02 0.30 Group 13 13.9 12.7 3.0 2.1 0.11 0.30 Group 14 52.6 20.62.9 0.82 0.69 0.70 Group 15 68 27.7 2.9 0.68 0.68 0.85 Group 16 13.9 212.4 0.79 0.46 0.72 Group 17 52 14.9 3.5 2.12 0.22 0.44

TABLE VI MEAN NORMALIZED INTENSITY VALUES (IN LOG FORMAT) OF GENES FORH9 EMBRYONIC STEM CELLS DERIVED DEFINITIVE ENDODERM STAGE CELLS CULTUREDON EITHER MATRIGEL ™ OR MEFS +/− WNT-3A. DE DE DE treatment treatment intreatment in low low serum + in low Benjamini serum + AA + Wnt- serum +and AA on 3A on AA on Hochberg- Gene Title MATRIGEL MATRIGEL MEFSP-value ESTs −4.82708 2.63968 −4.26995 8.62E−05microfibrillar-associated protein 4 0.063791 5.16358 −0.60091 3.48E−03Homo sapiens, alpha-1 (VI) collagen −3.66187 2.36459 −2.26934 1.45E−04ESTs −3.43712 2.14508 −2.6475 0.00E+00 Homo sapiens cystatin SN (CST1),0.072931 7.53908 4.63955 4.46E−03 mRNA. /PROD = cystatin SN /FL =gb:J03870.1 gb:NM_001898.1 solute carrier family 16 (monocarboxylic−0.066405 5.23572 0.279005 4.27E−04 acid transporters), member 3 /FL =gb:U81800.1 gb:NM_004207.1 Homo sapiens fibroblast growth factor−0.894644 5.75417 2.4872 4.93E−03 17 (FGF17), mRNA. /PROD = fibroblastgrowth factor 17 /FL = gb:NM_003867.1 gb:AB009249.1 Human link proteinmRNA, complete −1.93991 3.31348 −1.26346 1.14E−02 cds. /PROD = linkprotein /FL = gb:NM_001884.1 gb:U43328.1 Homo sapiens solute carrierfamily 16 0.710321 6.12971 1.72403 0.00E+00 (monocarboxylic acidtransporters), member 3 (SLC16A3), mRNA. /PROD = solute carrier family16 (monocarboxylic acidtransporters), member 3 /FL = gb:U81800.1gb:NM_004207.1 Homo sapiens apolipoprotein A-I −1.47073 5.37558 2.468915.07E−03 (APOA1), mRNA. /PROD = apolipoprotein- I precursor /FL =gb:M27875.1 gb:M11791.1 gb:NM_000039.1 gb:BC005380.1 Homo sapienscytidine deaminase −3.89129 2.05822 −1.67035 2.23E−03 (CDA), mRNA. /PROD= cytidine deaminase /FL = gb:L27943.1 gb:NM_001785.1 ESTs, Moderatelysimilar to JE0284 −2.37712 5.75671 4.22227 2.56E−02 Mm-1 cell derivedtransplantability- associated protein 1b (H. sapiens) ESTs −0.047165.28231 0.966974 0.00E+00 glycophorin B (includes Ss blood group)−2.85201 3.32812 −0.12969 1.45E−04 Homo sapiens homeobox protein−4.42042 3.55326 1.89424 2.50E−02 goosecoid mRNA, complete cds. /PROD =homeobox protein goosecoid /FL = gb:AY177407.1 gb:NM_173849.1 MCP-1 =monocyte chemotactic protein −2.27571 5.13499 2.95543 2.92E−02 (human,aortic endothelial cells, mRNA, 661 nt). /PROD = MCP-1 Homo sapiensMix-like homeobox −1.54648 4.47601 0.921971 2.01E−02 protein 1 (MILD1)mRNA, complete cds. /PROD = Mix-like homeobox protein 1 /FL =gb:AF211891.1 ESTs −4.93603 2.17917 −0.23735 1.12E−04 Homo sapienslumican (LUM), mRNA. −4.05726 3.21064 0.948822 3.39E−02 /PROD = lumican/FL = gb:NM_002345.1 gb:U18728.1 gb:U21128.1 Homo sapiens HNF-3beta mRNAfor −2.71785 4.68666 2.82506 3.71E−02 hepatocyte nuclear factor-3 beta,complete cds. /PROD = hepatocyte nuclear factor-3 beta /FL =gb:AB028021.1 gb:NM_021784.1 Homo sapiens reserved (KCNK12), −0.4687456.28184 3.77969 1.97E−02 mRNA. /PROD = tandem pore domain potassiumchannel THIK-2 /FL = gb:NM_022055.1 gb:AF287302.1 Homo sapiensatrophin-1 interacting −4.30828 1.80825 −1.32021 9.63E−03 protein 1;activin receptor interacting rotein 1 (KIAA0705), mRNA. /PROD =atrophin-1 interacting rotein 1; activinreceptor interacting protein 1/FL = gb:NM_012301.1 gb:AF038563.1 ESTs −2.33636 2.25176 −2.321245.26E−04 Homo sapiens glutamate decarboxylase −2.424 2.31908 −1.879654.07E−04 1 (brain, 67 kD) (GAD1), transcript variant GAD25, mRNA. /PROD= glutamate decarboxylase 1, isoform GAD25 /FL = gb:NM_013445.1gb:AF178853.1 gb:BC002815.1 Homo sapiens cardiac ventricular −0.5497284.89072 1.4377 2.99E−03 troponin C mRNA, complete cds. /PROD = cardiacventricular troponin C /FL = gb:NM_003280.1 gb:AF020769.1 ESTs −2.895543.42817 0.926036 2.62E−02 Homo sapiens fibroblast growth factor 8−4.32791 2.19561 0.015827 5.91E−03 (androgen-induced) (FGF8), mRNA./PROD = fibroblast growth factor 8 (androgen-induced) /FL = gb:U36223.1gb:U46212.1 gb:NM_006119.1 ESTs −3.09818 1.66254 −2.20564 8.62E−05 Homosapiens haptoglobin-related −2.6068 2.38009 −1.19632 6.95E−03 protein(HPR), mRNA. /PROD = haptoglobin-related protein /FL = gb:NM_020995.1collagen, type VI, alpha 1 −1.418 3.85952 0.604245 1.21E−02 Human (clone8B1) Br-cadherin mRNA, −2.17941 2.59894 −1.02624 1.24E−02 complete cds./PROD = Br-cadherin /FL = gb:L34057.1 gb:L33477.1 gb:NM_004061.1 ESTs−1.40092 2.57297 −1.82509 1.45E−04 Homo sapiens cystatin SA (CST2),−0.102178 5.21645 2.1671 2.40E−02 mRNA. /PROD = cystatin SA /FL =gb:NM_001322.1 Human mRNA for apolipoprotein AI (apo 0.215086 5.511092.49684 9.57E−03 AI) = . /PROD = preproapolipoprotein AI Homo sapiensMLL septin-like fusion −3.29221 1.70902 −1.49951 2.25E−03 MSF), mRNA./PROD = MLL septin-like fusion /FL = gb:AF123052.1 gb:NM_006640.1 Homosapiens cystatin S (CST4), 0.92448 6.48842 3.87036 7.20E−03 mRNA. /PROD= cystatin S /FL = gb:NM_001899.1 Homo sapiens phorbolin-like protein−1.11883 4.73391 2.40782 7.86E−03 MDS019 (MDS019), mRNA. /PROD =phorbolin-like protein MDS019 /FL = gb:AF182420.1 gb:NM_021822.1 Homosapiens apolipoprotein A-II −1.03333 5.80468 4.46856 3.23E−02 (APOA2),mRNA. /PROD = apolipoprotein A-II precursor /FL = gb:M29882.1gb:NM_001643.1 gb:BC005282.1 ESTs −1.55475 3.48278 0.420447 2.50E−02Homo sapiens glutamate decarboxylase −3.86752 1.56384 −1.08675 2.52E−021 (brain, 67 kD) (GAD1), transcript variant GAD67, mRNA. /PROD =glutamate decarboxylase 1, isoform GAD67 /FL = gb:NM_000817.1gb:M81883.1 gb:L16888.1 ESTs −0.731491 5.43249 3.52168 1.60E−02 ESTs−2.03591 3.38924 0.760984 2.34E−03 Homo sapiens retinoid X receptor,−2.37496 2.62934 −0.32035 8.83E−04 gamma (RXRG), mRNA. /PROD = retinoidX receptor, gamma /FL = gb:NM_006917.1 gb:U38480.1 ESTs −0.6485524.30576 1.43266 2.19E−03 Homo sapiens cDNA FLJ11550 fis, −1.222285.37746 4.21644 1.68E−02 clone HEMBA1002970 ESTs −1.782 3.50391 1.05011.85E−02 Homo sapiens haptoglobin (HP), mRNA. −1.10114 3.5449 0.4770278.13E−03 /PROD = haptoglobin /FL = gb:K00422.1 gb:L29394.1gb:NM_005143.1 Homo sapiens hypothetical protein −0.431989 5.144973.02045 2.62E−02 FLJ10970 (FLJ10970), mRNA. /PROD = hypothetical proteinFLJ10970 /FL = gb:NM_018286.1 Homo sapiens beta-site APP cleaving−2.0354 3.70648 1.75385 8.00E−03 enzyme (BACE) mRNA, complete cds. /PROD= beta-site APP cleaving enzyme /FL = gb:AF200343.1 gb:AF204943.1gb:AF190725.1 gb:AF201468.1 gb:NM_012104.1 Homo sapiens hypotheticalprotein 1.36784 6.82571 4.5979 2.10E−02 FLJ22252 similar to SRY-boxcontaining gene 17 (FLJ22252), mRNA. /PROD = hypothetical proteinFLJ22252 similar to SRY-boxcontaining gene 17 /FL = gb:NM_022454.1Cluster Incl. AB028021:Homo sapiens −1.5339 5.12418 4.11704 4.47E−02HNF-3beta mRNA for hepatocyte nuclear factor-3 beta, complete cds /cds =(196, 1569) /gb = AB028021 /gi = 4958949 /ug = Hs.155651 /len = 1944Homo sapiens gastrin-releasing peptide −2.74071 2.70077 0.5097572.49E−04 (GRP), mRNA. /PROD = gastrin-releasing peptide /FL =gb:NM_002091.1 gb:K02054.1 gb:BC004488.1 Homo sapiens sema domain, seven−1.53335 3.78503 1.48732 4.71E−02 thrombospondin repeats (type 1 andtype 1-like), transmembrane domain (TM) and short cytoplasmic domain,(semaphorin) 5A (SEMA5A), mRNA. /PROD = sema domain, seventhrombospondin repeats (type1 and type 1-like), transmem Homo sapiensmRNA; cDNA −0.835182 5.22406 3.69882 2.08E−02 DKFZp586J0624 (from cloneDKFZp586J0624); complete cds. /PROD = hypothetical protein /FL =gb:AF215636.1 gb:NM_014585.1 gb:AF231121.1 gb:AF226614.1 gb:AL 136944.1Human mRNA for alpha-1 type II −2.8736 2.155 −0.38021 5.70E−03 collagen.ho GDP dissociation inhibitor (GDI) −1.54385 1.7147 −2.58241 1.71E−02alpha neuropilin 1 /FL = gb:AF016050.1 −1.62253 1.95432 −1.9667 8.83E−04gb:NM_003873.1 gb:AF018956.1 Human DNA sequence from clone RP1- −3.723131.68755 −0.37308 1.38E−03 181C24 on chromosome 6p11.1-12.2. Contains the3 end of the BMP5 gene for bone morphogenetic protein 5, ESTs, STSs andGSSs /FL = gb:M60314.1 gb:NM_021073.1 myristoylated alanine-rich proteinkinase −0.71724 3.51728 0.335725 4.29E−03 C substrate (MARCKS, 80K-L)/FL = gb:M68956.1 gb:D10522.1 gb:NM_002356.4 hypothetical proteinFLJ23403 −1.45618 1.81423 −2.31327 1.20E−02 /FL = gb:NM_022068.1hepatocyte nuclear factor 4, alpha −4.26574 1.7879 0.445241 3.25E−02Homo sapiens cell adhesion molecule −0.541188 2.1751 −2.5002 1.16E−03with homology to L1CAM (close homologue of L1) (CHL1), mRNA. /PROD =cell adhesion molecule with homology to L1CAM(close homologue of L1) /FL= gb:AF002246.1 gb:NM_006614.1 matrix metalloproteinase 14 (membrane-−2.05734 2.36236 −0.5185 1.66E−02 inserted) /FL = gb:U41078.1gb:NM_004995.2 Homo sapiens glycophorin B (includes −0.947308 3.260890.180293 4.83E−04 Ss blood group) (GYPB), mRNA. /PROD = glycophorin Bprecursor /FL = gb:J02982.1 gb:NM_002100.2 WAS protein family, member 2−2.18746 1.99129 −1.05968 4.00E−03 /FL = gb:NM_006990.1 gb:AB026542.1Homo sapiens frizzled-related protein 0.56502 5.7261 3.67629 1.75E−02(FRZB), mRNA. /PROD = frizzled-related protein /FL = gb:U24163.1gb:U68057.1 gb:NM_001463.1 gb:U91903.1 Homo sapiens glutamatedecarboxylase −1.68495 2.27067 −0.96944 8.02E−03 (brain, 67 kD) (GAD),transcript variant GAD25, mRNA. /PROD = glutamate decarboxylase 1,isoform GAD25 /FL = gb:NM_013445.1 gb:AF178853.1 gb:BC002815.1 ESTs0.82766 5.93144 3.91314 4.15E−02 Homo sapiens clone 23736 mRNA −0.0471825.79006 4.50744 1.74E−02 sequence Homo sapiens glycophorin E (GYPE),−2.01601 1.79002 −1.50134 1.97E−02 mRNA. /PROD = glycophorin E /FL =gb:NM_002102.1 gb:M29610.1 ESTs 1.06767 5.63319 3.12487 2.00E−02 ESTs−1.41162 2.5396 −0.57029 1.29E−02 Human Fritz mRNA, complete cds.0.436589 5.69814 3.91514 1.99E−02 /PROD = Fritz /FL = gb:U24163.1gb:U68057.1 gb:NM_001463.1 gb:U91903.1 Homo sapiens, clone MGC:4655,2.3772 5.9184 2.47596 1.20E−02 mRNA, complete cds. /PROD = Unknown(protein for MGC:4655) /FL = gb:BC004908.1 KIAA0878 protein /FL =gb:NM_014899.1 1.1189 6.41747 4.78882 2.01E−02 gb:AB020685.1 Homosapiens sema domain, −0.785987 3.69668 1.27624 2.10E−02 immunoglobulindomain (Ig), short basic domain, secreted, (semaphorin) 3E (SEMA3E),mRNA. /PROD = sema domain, immunoglobulin domain (Ig), shortbasicdomain, secreted, (semaphorin) 3E /FL = gb:NM_012431.1 gb:AB002329.1ESTs 1.48084 6.59709 4.81395 1.36E−02 noggin /FL = gb:NM_005450.1−1.63627 3.28161 1.32958 2.53E−02 Homo sapiens hypothetical protein−0.904749 3.35854 0.755319 1.12E−04 FLJ11316 (FLJ11316), mRNA. /PROD =hypothetical protein FLJ11316 /FL = gb:NM_018388.1 Homo sapiensangiopoietin 2 (ANGPT2), −2.93044 2.23779 0.59685 3.43E−02 RNA. /PROD =angiopoietin 2 /FL = gb:AB009865.1 gb:AF004327.1 gb:NM_001147.1 Homosapiens matrix metalloproteinase −0.723489 2.97262 −0.09689 5.44E−03 14(membrane-inserted) (MMP14), mRNA. /PROD = matrix metalloproteinase 14preproprotein /FL = gb:U41078.1 gb:NM_004995.2 G protein-coupledreceptor 1.50709 6.65228 5.05327 2.00E−02 collagen, type IX, alpha 21.27026 5.4659 2.93507 3.19E−03 /FL = gb:NM_001852.1 ESTs 0.5216383.93176 0.620223 0.00E+00 KIAA1462 protein −3.84563 1.65452 0.4370643.27E−02 Homo sapiens cartilage linking protein 1 −1.31515 2.27271−0.80521 2.22E−02 (CRTL1), mRNA. /PROD = cartilage linking protein 1 /FL= gb:NM_001884.1 gb:U43328.1 Homo sapiens solute carrier family 210.711428 4.89808 2.43304 6.84E−03 (prostaglandin transporter), member 2(SLC21A2), mRNA. /PROD = solute carrier family 21(prostaglandintransporter), member 2 0.307173 4.78515 2.65653 1.72E−02/FL = gb:U70867.1 gb:NM_005630.1 ESTs6-phosphofructo-2-kinasefructose-2,6- −0.242865 3.97929 1.59985 5.28E−03biphosphatase 4 Homo sapiens dual specificity 0.953857 5.82811 4.121594.00E−03 phosphatase 4 (DUSP4), mRNA. /PROD = dual specificityphosphatase 4 /FL = gb:NM_001394.2 gb:BC002671.1 gb:U48807.1 gb:U21108.1ESTs, Weakly similar to T00331 −1.57372 2.70797 0.443622 6.74E−03hypothetical protein KIAA0555 (H. sapiens) ESTs −3.57414 3.15167 3.371988.82E−03 ESTs, Highly similar to IHH_HUMAN −0.653989 3.22059 0.5905335.18E−03 INDIAN HEDGEHOG PROTEIN PRECURSOR (H. sapiens) STs, Weaklysimilar to FC 2 MOUSE 0.494192 5.22522 3.48031 1.97E−02 LOW AFFINITYIMMUNOGLOBULIN EPSILON FC RECEPTOR (M. musculus) homeo box HB9 /FL =gb:NM_005515.1 −1.65563 3.2238 1.63092 5.58E−03 Homo sapiensarylsulfatase E 0.283004 4.95903 3.18424 9.22E−03 (chondrodysplasiapunctata 1) (ARSE), mRNA. /PROD = arylsulfatase E precursor /FL =gb:X83573.1 gb:NM_000047.1 ESTs −0.05909 3.0455 −0.29817 1.47E−02 Homosapiens hypothetical protein −1.48452 1.97473 −1.00431 5.72E−03 FLJ23403(FLJ23403), mRNA. /PROD = hypothetical protein FLJ23403 /FL =gb:NM_022068.1 ESTs −0.182403 3.01548 −0.18954 3.72E−03 hypotheticalprotein FLJ23091 0.323388 5.25192 3.77987 3.57E−02 Human dipeptidylpeptidase IV (CD26) −3.61145 0.760585 −1.25595 3.07E−02 mRNA, completecds. /PROD = dipeptidyl peptidase IV /FL = gb:M74777.1 hypotheticalprotein FLJ21032 0.355672 4.67756 2.61753 4.59E−02 Homo sapiens Kellblood group (KEL), −2.20519 1.89439 −0.38393 1.91E−02 mRNA. /PROD = Kellblood group antigen /FL = gb:BC003135.1 gb:NM_000420.1 0.7481 4.271692.97E−03 splicing factor, arginineserine-rich 5 5.68934 Human prostaticsecretory protein 57 −3.01313 1.46338 −0.40767 1.97E−02 mRNA, completecds. /PROD = PSP57 /FL = gb:U22178.1 Homo sapiens KIAA0878 protein2.0265 7.00937 5.65368 2.85E−02 (KIAA0878), mRNA. /PROD = KIAA0878protein /FL = gb:NM_014899.1 gb:AB020685.1 Homo sapiens cryptic mRNA,complete 0.104874 2.87319 −0.67353 1.61E−03 cds. /PROD = cryptic /FL =gb:AF312769.1 Homo sapiens cDNA FLJ13221 fis, 0.355743 3.98782 1.309631.38E−03 clone NT2RP4002075 phorbolin-like protein MDS019 −1.117562.83853 0.503523 1.59E−02 Homo sapiens mRNA for KIAA1409 0.368334 2.8009−1.03191 1.78E−02 protein, partial cds. /PROD = KIAA1409 rotein Homosapiens mRNA; cDNA −2.63427 1.64513 −0.3056 2.52E−02 DKFZp434D0818 (fromclone DKFZp434D0818) ESTs 0.35393 5.14775 3.74875 3.16E−02 Homo sapiensTBX3-iso protein (TBX3- −2.34566 2.45238 1.07038 1.31E−02 iso), mRNA./PROD = TBX3-iso protein /FL = gb:NM_016569.1 gb:AF216750.1 Homo sapienschromosome 19, cosmid −0.258871 3.0636 0.223926 1.58E−03 R31181 Homosapiens mRNA for GATA-6, 2.21862 6.8609 5.34263 4.29E−02 complete cds./PROD = GATA-6 /FL = gb:U66075.1 gb:NM_005257.1 gb:D87811.1 Homo sapiensankyrin-like with −1.10879 3.93484 2.81939 1.29E−02 transmembranedomains 1 (ANKTM1), mRNA G protein-coupled receptor 49 0.265509 4.462572.50537 2.19E−02 Homo sapiens growth differentiation 1.67253 5.349442.87486 8.62E−05 factor 3 (GDF3), mRNA. /PROD = growth differentiationfactor 3 precursor /FL = gb:NM_020634.1 gb:AF263538.1 Human (cloneHSY3RR) neuropeptide Y 1.77461 6.70301 5.47995 3.00E−02 receptor (NPYR)mRNA, complete cds. /PROD = neuropeptide Y receptor /FL = gb:L06797.1gb:NM_003467.1 gb:AF025375.1 gb:AF147204.1 gb:M99293.1 gb:L01639.1 Homosapiens type VI collagen alpha 2 1.7011 5.33126 2.84458 5.91E−03 chainprecursor (COL6A2) mRNA, complete cds, alternatively spliced. /PROD =type VI collagen alpha 2 chain precursor /FL = gb:AY029208.1 ESTs−0.349726 3.29119 0.824929 4.11E−03 ESTs −0.903317 1.89777 −1.364296.84E−03 Homo sapiens hypothetical protein 2.60483 7.10633 5.552421.41E−02 FLJ10718 (FLJ10718), mRNA. /PROD = hypothetical proteinFLJ10718 FL = gb:NM_018192.1 Homo sapiens Rho GTPase activating −1.103891.83053 −1.28521 1.28E−02 protein 6 (ARHGAP6), transcript variant 2,mRNA. /PROD = Rho GTPase activating protein 6 isoform 2 /FL =gb:AF022212.2 gb:NM_001174.2 2.41135 7.29563 6.14284 2.51E−02stanniocalcin 1 /FL = gb:U25997.1 gb:NM_003155.1 gb:U46768.1 Humanglycophorin HeP2 mRNA, partial −0.843493 2.71108 0.233547 2.98E−04 cds./PROD = glycophorin HeP2 Homo sapiens cDNA FLJ12993 fis, 0.1472594.12241 2.06949 6.84E−03 clone NT2RP3000197 Homo sapiens presenilinstabilization −0.86173 2.85614 0.56745 6.09E−03 factor b (PSF) mRNA,complete cds; alternatively spliced. /PROD = presenilin stabilizationfactor b /FL = gb:AY113699.1 Homo sapiens glycophorin Erik STA −1.193622.17108 −0.45439 1.45E−04 (GPErik) gene complete cds. /PROD =glycophorin Erik (STA) /FL = gb:U00178.1 bromodomain and PHD finger−2.8472 1.75573 0.369397 8.62E−05 containing, 3 ESTs 0.784344 4.741042.71543 7.55E−03 ESTs −1.26251 3.4693 2.27614 3.80E−03 ESTs −1.717131.23763 −1.71122 2.99E−02 Homo sapiens microsomal glutathione 2.062336.81536 5.6918 3.79E−02 S-transferase 2 (MGST2), mRNA. /PROD =microsomal glutathione S- transferase 2 /FL = gb:NM_002413.1 gb:U77604.1Homo sapiens eomesodermin (Xenopus 2.65926 6.71627 4.89839 2.76E−02laevis) homolog (EOMES), mRNA. /PROD = eomesodermin (Xenopus laevis)homolog /FL = gb:AB031038.1 gb:NM_005442.1 Homo sapiens mRNA for MSX-2,0.407211 4.11145 1.94529 1.35E−02 complete cds. /PROD = MSX-2 /FL =gb:D89377.1 Homo sapiens apolipoprotein A-II −1.10237 5.10066 4.758991.34E−02 (APOA2), mRNA. /PROD = apolipoprotein A-II precursor /FL =gb:M29882.1 gb:NM_001643.1 gb:BC005282.1 Homo sapiens adenylate cyclase8 −1.3408 1.12773 −2.26149 1.07E−02 (brain) (ADCY8), mRNA. /PROD =adenylate cyclase 8 /FL = gb:NM_001115.1 Homo sapiensglucose-6-phosphate −0.193516 3.03064 0.407738 1.38E−03 transporter(G6PT) gene, G6PT-Dt allele, complete cds Homo sapiens glutathioneS-transferase −3.10645 1.11704 −0.4221 5.37E−04 A2 (GSTA2), mRNA. /PROD= glutathione S-transferase A2 /FL = gb:BC002895.1 gb:M25627.1gb:M16594.1 gb:M14777.1 gb:M15872.1 gb:M21758.1 gb:NM_000846.1 Homosapiens sodium dependent −0.397133 2.78341 0.231961 1.11E−02 phosphatetransporter isoform NaPi-IIb mRNA, complete cds. /PROD = sodiumdependent phosphate transporter isoformNaPi-IIb /FL = gb:AF111856.1gb:NM_006424.1 gb:AF146796.1 ESTs 2.77413 7.19192 5.906 3.17E−02 Homosapiens partial LHX9 gene for −2.2486 1.64952 −0.13424 7.89E−03LIM-homeobox 9, 3UTR. Homo sapiens LYST-interacting protein −0.6821492.34271 −0.31253 9.87E−03 LIP3 mRNA, partial cds. /PROD = LYST-interacting protein LIP3 putative 47 kDa protein −0.123937 3.29141.05247 3.72E−03 Homo sapiens protein S (alpha) 0.577604 4.00035 1.78837.89E−03 (PROS1), mRNA. /PROD = protein S (alpha) /FL = gb:M15036.1gb:NM_000313.1 ESTs −1.63711 0.915477 −2.15328 1.83E−02 protocadherin 101.76718 5.1503 2.98569 2.52E−02 KIAA1511 protein 0.620278 3.868861.57543 1.86E−03 Homo sapiens cDNA FLJ13221 fis, 0.282785 3.441781.08178 1.31E−03 lone NT2RP4002075 Homo sapiens fibroblast growth factor−1.72385 2.26674 0.747239 6.86E−03 receptor 4, soluble-form splicevariant (FGFR4) mRNA, complete cds. /PROD = fibroblast growth factorreceptor 4, soluble-form splice variant −1.36729 2.60456 1.082041.58E−02 /FL = gb:NM_022963.1 gb:AF202063.1 X75208/FEATURE = cds/DEFINITION = HSPTKR H. sapiens HEK2 mRNA for protein tyrosine kinasereceptor Homo sapiens chemokine receptor 2.30557 6.72969 5.669333.31E−02 CXCR4 mRNA, complete cds. /PROD = chemokine receptor CXCR4 /FL= gb:AF348491.1 KIAA1415 protein 0.185854 3.70346 1.74255 7.78E−04 ESTs−0.048335 3.11786 0.836448 1.29E−02 Homo sapiens stanniocalcin 1 (STC1),2.39435 6.55737 5.28007 2.19E−02 mRNA. /PROD = stanniocalcin 1 /FL =gb:U25997.1 gb:NM_003155.1 gb:U46768.1 Homo sapiens high mobility group1.04695 3.2786 0.095286 2.11E−02 protein-R mRNA, complete cds. /PROD =high mobility group protein-R /FL = gb:AF176039.1 ESTs, Moderatelysimilar to −1.10358 1.66331 −0.98371 3.37E−02 ALU4_HUMAN ALU SUBFAMILYSB2 SEQUENCE CONTAMINATION WARNING ENTRY (H. sapiens) Homo sapiensarrestin, beta 1 (ARRB1), −0.158844 2.41791 −0.40915 1.12E−02 transcriptvariant 1, mRNA. /PROD = arrestin beta 1, isoform A /FL = gb:BC003636.1gb:AF084040.1 gb:NM_004041.2 ESTs −1.34399 1.80279 −0.44529 1.60E−03Human mRNA for pro-alpha 1 (II) 0.85982 4.1935 2.15657 1.09E−02 collagen3end C-term. triple helical and C-terminal non-helical domain. PROD =pro-alpha 1 (II) collagen (313 AA; AA 975-271c) /FL = gb:NM_001844.2Homo sapiens hypothetical protein 1.47773 4.42479 2.00326 1.16E−03DKFZp564B052 (DKFZp564B052), mRNA. /PROD = hypothetical proteinDKFZp564B052 /FL = gb:NM_030820.1 testis enhanced gene transcript (BAX−0.695228 2.99328 1.31679 1.17E−03 inhibitor 1) Homo sapiensfasciculation and 1.86433 4.71354 2.20315 8.62E−05 elongation proteinzeta 1 (zygin I) (FEZ1), transcript variant 1, mRNA. /PROD = zygin 1,isoform 1 /FL = gb:U60060.1 gb:U69139.1 gb:NM_005103.2 Homo sapiensmatrix metalloproteinase −0.298504 3.44582 1.8336 4.80E−03 15(membrane-inserted) (MMP15), mRNA. /PROD = matrix metalloproteinase 15preproprotein /FL = gb:D86331.1 gb:NM_002428.1 heparan sulfateproteoglycan 2 −2.53327 0.924726 −0.97212 4.31E−02 (perlecan) /FL =gb:M85289.1 gb:NM_005529.2 Homo sapiens dickkopf (Xenopus laevis)0.981469 3.70716 1.09014 1.76E−03 homolog 1 (DKK1), mRNA. /PROD =dickkopf (Xenopus laevis) homolog 1 /FL = gb:AF177394.1 gb:NM_012242.1gb:AF127563.1 ESTs −0.801487 3.37342 2.21393 1.22E−02 ESTs −2.576261.40231 0.050493 6.60E−03 ESTs 0.499579 4.10541 2.39934 4.29E−03 Homosapiens bone morphogenetic 2.04506 6.19114 5.02927 4.98E−02 protein 2(BMP2), mRNA. /PROD = bone morphogenetic protein 2 precursor /FL =gb:NM_001200.1 Human extracellular matrix protein 1 0.634778 3.658291.37564 6.11E−04 (ECM1) mRNA, complete cds. /PROD = extracellular matrixprotein 1 /FL = gb:NM_004425.2 gb:U65932.1 b:U68186.1 Homo sapiensnuclear receptor −1.92783 2.32456 1.27907 2.99E−02 subfamily 0, group B,member 1 (NR0B1), mRNA. /PROD = adrenal hypoplasia protein /FL =gb:NM_000475.2 Homo sapiens cDNA: FLJ23067 fis, −2.25626 1.959870.882498 6.62E−03 clone LNG04993. Homo sapiens ADP-ribosylation factor4- 1.63466 5.70351 4.48037 1.90E−02 like (ARF4L), mRNA. /PROD = ADP-ribosylation factor 4-like /FL = gb:U25771.1 gb:L38490.1 gb:NM_001661.1gb:BC000043.1 Homo sapiens HT016 mRNA, complete 0.218762 3.97514 2.452223.37E−02 cds. /PROD = HT016 /FL = gb:AF225426.1 Homo sapiens,tropomodulin, clone 0.348796 3.54199 1.46427 1.29E−03 MGC:3643, mRNA,complete cds. /PROD = tropomodulin /FL = gb:NM_003275.1 gb:M77016.1gb:BC002660.1 Homo sapiens hypothetical protein 0.903825 4.65366 3.13492.48E−02 FLJ22471 (FLJ22471), mRNA. /PROD = hypothetical proteinFLJ22471 /FL = gb:NM_025140.1 ESTs −0.120583 2.94229 0.738304 7.20E−03ESTs, Moderately similar to JC4969 pig- −2.39713 0.906454 −1.044951.69E−02 c protein (H. sapiens) Homo sapiens LIM homeobox protein 1−0.77166 2.27746 0.085559 4.26E−02 (LHX1), mRNA. /PROD = LIM homeoboxprotein 1 /FL = gb:NM_005568.1 gb:U14755.1 Homo sapiens, hypotheticalprotein −2.43105 0.646883 −1.50617 8.27E−03 MGC2865, clone MGC:20246IMAGE:4635389, mRNA, complete cds. /FL = gb:BC016043.1 ESTs −2.015232.16773 1.12403 1.99E−02 Homo sapiens oxoglutarate −0.032595 2.3997−0.38533 1.45E−02 dehydrogenase (lipoamide) (OGDH), mRNA. /PROD =oxoglutarate dehydrogenase (lipoamide) /FL = gb:D10523.1 gb:BC004964.1ESTs, Weakly similar to T32252 gb:NM_002541.1 ESTs, Weakly similar toT32252 2.20966 5.35974 3.33255 1.12E−04 hypothetical protein T15B7.2-Caenorhabditis elegans (C. elegans) Homo sapiens, cleft lip and palate1.31578 3.52357 0.556735 2.92E−03 associated transmembrane protein 1,clone MGC: 10593, mRNA, complete cds. /PROD = cleft lip and palateassociated transmembraneprotein 1 /FL = gb:BC004865.1 Homo sapiens cloneTUA8 Cri-du-chat 0.09218 3.37126 1.49746 6.46E−03 region mRNA Homosapiens transcriptional activator of 0.666391 4.50481 3.22532 5.80E−03the c-fos promoter (CROC4), mRNA. /PROD = transcriptional activator ofthe c- fos promoter /FL = gb:NM_006365.1 gb:U49857.1 Homo sapienshypothetical protein −1.38519 2.23626 0.763963 7.36E−03 FLJ21195 similarto protein related to DAC and cerberus (FLJ21195), mRNA. /PROD =hypothetical protein FLJ21195 similar to proteinrelated to DAC andcerberus /FL = gb:NM_022469.1 ras homolog gene family, member B−0.123101 2.52093 0.071885 1.86E−02 /FL = gb:AF498971.1 gb:NM_004040.1cartilage linking protein 1 0.879449 3.47789 0.987243 1.03E−03 Homosapiens BCL2adenovirus E1B 3.30506 6.91803 5.44746 1.79E−03 19kD-interacting protein 3 (BNIP3) mRNA, complete cds. /PROD =BCL2adenovirus E1B 19 kD- interacting protein 3 /FL = gb:AF002697.1gb:NM_004052.2 gb:U15174.1 Homo sapiens polycythemia rubra vera −1.196292.40987 0.93741 9.40E−03 1; cell surface receptor (PRV1), mRNA. /PROD =polycythemia rubra vera 1; cell surfacereceptor /FL = gb:NM_020406.1b:AF146747.1 Homo sapiens hypothetical protein −0.044112 3.42017 1.806883.77E−03 FLJ11560 (FLJ11560), mRNA. /PROD = hypothetical proteinFLJ11560 /FL = gb:NM_025182.1 plexin A2 0.930527 4.36203 2.741671.52E−02 Homo sapiens mRNA; cDNA 1.37193 4.24502 2.08129 5.37E−04DKFZp547H236 (from clone DKFZp547H236). /PROD = hypothetical proteinHomo sapiens cystatin C (amyloid 2.08601 5.30525 3.51965 2.20E−03angiopathy and cerebral hemorrhage) (CST3), mRNA. /PROD = cystatin C(amyloid angiopathy and cerebralhemorrhage) /FL = gb:NM_000099.1 ESTs,Moderately similar to NFY-C −0.749407 2.00148 −0.24268 2.34E−03 (H.sapiens) ESTs 2.54243 5.75201 3.97307 2.60E−02 Homo sapienssialyltransferase (STHM), −0.68709 2.89586 1.50348 3.70E−02 mRNA. /PROD= sialyltransferase /FL = gb:U14550.1 gb:NM_006456.1 Homo sapiens SG2NAbeta isoform −1.15566 1.13989 −1.53439 1.55E−02 mRNA, partial cds. /PROD= SG2NA beta isoform Homo sapiens hypothetical protein 1.49091 4.801383.15205 2.63E−02 FLJ12838 (FLJ12838), mRNA. /PROD = hypothetical proteinFLJ12838 /FL = gb:NM_024641.1 Homo sapiens solute carrier 0.4037062.12385 −1.10043 1.78E−02 (SLC25A18) mRNA, complete cds; nuclear genefor mitochondrial product. /PROD = solute carrier /FL = gb:AY008285.1KIAA0346 protein −1.34736 2.49605 1.41497 3.90E−03 Human clone 23826mRNA sequence 1.80782 4.07112 1.44315 2.28E−04 Homo sapiens receptortyrosine kinase- 1.53333 4.73839 3.05612 1.63E−02 like orphan receptor 2(ROR2), mRNA. PROD = receptor tyrosine kinase-like orphan receptor 2 /FL= gb:M97639.1 gb:NM_004560.1 Homo sapiens MYC-associated zinc −0.0555692.67075 0.511292 2.63E−02 finger protein (purine-binding transcriptionfactor) (MAZ), mRNA. /PROD = MYC-associated zinc fingerprotein(purine-binding transcription factor) /FL = gb:D85131.1gb:NM_002383.1 Homo sapiens cDNA FLJ30081 fis, 0.677905 3.33728 1.124991.12E−04 clone BGGI12000693, weakly similar to POLYHOMEOTIC-PROXIMALCHROMATIN PROTEIN. Homo sapiens elongation of very long 0.099979 3.233721.50802 2.59E−03 chain fatty acids (FEN1Elo2, SUR4Elo3, yeast)-like 2(ELOVL2), mRNA. /PROD = elongation of very long chain fattyacids(FEN1Elo2, SUR4Elo3, yeast)-like 2 /FL = gb:NM_017770.1 Homosapiens thyrotropin-releasing −1.65672 1.46994 −0.25839 3.02E−02 hormone(TRH), mRNA. /PROD = thyrotropin-releasing hormone /FL = gb:NM_007117.1ESTs, Weakly similar to A46302 PTB- 0.894051 4.21835 2.69535 1.57E−02associated splicing factor, long form (H. sapiens) Human MLC1emb genefor embryonic 1.3489 5.11354 4.03961 1.61E−02 myosin alkaline lightchain, promoter and exon 1 Homo sapiens microseminoprotein, −2.379041.3101 0.169915 2.46E−02 beta- (MSMB), mRNA. /PROD = microseminoprotein,beta- /FL = gb:NM_002443.1 Homo sapiens cDNA FLJ11390 fis, −1.129442.02889 0.364373 0.00E+00 clone HEMBA1000561, weakly similar to ZINCFINGER PROTEIN 91. ESTs, Weakly similar to ALU8_HUMAN 2.76608 6.079494.57766 1.93E−02 LU SUBF MILY SX SEQUENCE CONTAMINATION WARNING ENTRY(H. sapiens) Homo sapiens cDNA FLJ13810 fis, −2.92389 0.51764 −0.853228.08E−03 clone THYRO1000279 Homo sapiens, aminolevulinate, delta-,1.3099 4.44525 2.76918 7.69E−03 dehydratase, clone MGC:5057, mRNA,complete cds. /PROD = aminolevulinate, delta-, dehydratase /FL =gb:BC000977.1 gb:M13928.1 gb:NM_000031.1 Homo sapiens kidney-specific1.91929 5.08917 3.44968 6.91E−03 membrane protein NX-17 mRNA, completecds. /PROD = kidney-specific membrane protein NX-17 /FL = gb:AF229179.1Homo sapiens alpha 2,8- −1.10548 2.36359 1.05469 4.63E−03sialyltransferase mRNA, complete cds. /PROD = alpha2,8-sialyltransferase /FL = gb:L43494.1 gb:D26360.1 gb:L32867.1gb:NM_003034.1 tyrosine 3-monooxygenasetryptophan 5- 2.98486 6.179184.60758 8.01E−03 monooxygenase activation protein, eta polypeptide Homosapiens cardiac ankyrin repeat −0.002291 3.35645 1.98688 3.44E−02protein (CARP), mRNA. /PROD = cardiac ankyrin repeat protein /FL =gb:NM_014391.1 Homo sapiens porcupine (MG61), 1.51754 4.96311 3.681039.86E−03 mRNA. /PROD = porcupine /FL = gb:AF317059.1 gb:AF317058.1gb:NM_022825.1 collagen, type V, alpha 1 1.5875 3.98844 1.66569 1.17E−03/FL = gb:D90279.1 gb:NM_000093.1 gb:M76729.1 Homo sapiens forkhead boxF2 −1.54469 1.02305 −1.11517 6.95E−03 (FOXF2), mRNA. /PROD = forkheadbox F2 /FL = gb:U13220.1 gb:NM_001452.1 −1.92021 3.74378 2.772772.10E−02 PTPRF interacting protein, binding rotein 2 (liprin beta 2)Homo sapiens PRO1957 mRNA, 1.68941 5.30311 4.22705 1.43E−02 completecds. /PROD = PRO1957 /FL = gb:AF116676.1 Homo sapiens hypotheticalprotein 0.564613 5.98527 5.24692 2.85E−02 FLJ23514 (FLJ23514), mRNA./PROD = hypothetical protein FLJ23514 /FL = gb:NM_021827.1 Homo sapiensmRNA; cDNA −1.80897 1.69722 0.523009 2.47E−02 DKFZp586G2120 (from cloneDKFZp586G2120); complete cds. /PROD = hypothetical protein /FL =gb:AL136924.1 Cluster Incl. L37033:Human FK-506 0.615619 3.478 1.661321.54E−02 binding protein homologue (FKBP38) mRNA, complete cds /cds =(140,1207) /gb = L37033 /gi = 965469 /ug = Hs.173464 /len = 1613 ESTs−0.004009 3.63657 2.61352 4.29E−02 Homo sapiens apolipoprotein C-I3.24954 6.71073 5.50997 2.16E−02 (APOC1), mRNA. /PROD = apolipoproteinC-I precursor /FL = gb:NM_001645.2 ESTs 1.38635 4.93112 3.83641 1.39E−02Homo sapiens cDNA FLJ34035 fis, −3.58002 −0.48718 −2.00751 1.46E−02clone FCBBF2004788. Homo sapiens, clone IMAGE:3509274, 0.493686 2.949230.794513 1.82E−02 mRNA, partial cds Homo sapiens putative sterolreductase 1.66857 4.3767 2.47833 5.37E−04 SR-1 (TM7SF2) mRNA, completecds. /PROD = putative sterol reductase SR-1 /FL = gb:AF096304.1 nuclearfactor of kappa light polypeptide 2.91463 6.48938 5.47812 1.97E−02 geneenhancer in B-cells inhibitor, alpha /FL = gb:NM_020529.1 gb:BC002601.1gb:BC004983.1 gb:M69043.1 hypothetical protein FLJ12666 0.215767 3.102491.40459 1.91E−02 Homo sapiens ectodermal dysplasia 1, 0.796627 4.228953.07833 1.01E−02 anhidrotic (ED1), mRNA. PROD = ectodermal dysplasia 1,anhidrotic /FL = gb:AF060999.1 gb:NM_001399.1 gb:AF040628.1gb:AF061189.1 0.274378 3.59045 2.3278 2.64E−02 glycoprotein M6A /FL =gb:D49958.1 ESTs 1.04714 3.87608 2.12752 3.75E−02 ESTs −1.48489 1.371−0.35043 1.88E−03 ESTs, Weakly similar to KIAA1330 −0.925232 2.411491.18187 2.65E−03 protein (H. sapiens) putative 47 kDa protein 0.1564842.78771 0.861726 2.60E−02 Homo sapiens hypothetical protein −0.5956224.84785 3.94683 3.34E−02 FLJ32835 (FLJ32835), mRNA. /FL = gb:NM_152506.1Homo sapiens GS1999full mRNA, 0.137029 2.75189 0.849942 0.00E+00complete cds. /FL = gb:AB048286.1 Homo sapiens hexabrachion (tenascin3.96862 5.66572 2.85391 1.39E−02 C, cytotactin) (HXB), mRNA. /PROD =hexabrachion (tenascin C, cytotactin) /FL = gb:M55618.1 gb:NM_002160.1Homo sapiens Pig10 (PIG10) mRNA, 0.129454 2.86013 1.09765 1.16E−03complete cds. /PROD = Pig10 /FL = gb:AF059611.1 gb:AF010314.1gb:NM_003633.1 gb:BC000418.1 gb:AF005381.1 Homo sapiens annexin A6(ANXA6), 3.89026 6.43752 4.49981 8.81E−04 transcript variant 1, mRNA./PROD = annexin VI isoform 1 /FL = gb:D00510.1 gb:J03578.1gb:NM_001155.2 Homo sapiens c-mer proto-oncogene 1.78994 4.92901 3.616651.84E−02 tyrosine kinase (MERTK), mRNA. /PROD = c-mer proto-oncogenetyrosine kinase /FL = gb:NM_006343.1 gb:U08023.1 Homo sapienscaspase-like apoptosis 0.434199 5.04863 5.2167 2.46E−02 regulatoryprotein 2 (clarp) mRNA, alternatively spliced, complete cds. PROD =caspase-like apoptosis regulatory protein 2 /FL = gb:AF005775.1 Humanembryonic myosin alkali light 1.94171 5.24959 4.11475 2.11E−02 chain(MLC1) mRNA, complete cds. /FL = gb:M36172.1 gb:M24121.1 gb:NM_002476.1Homo sapiens phosphatidylinositol-4- 1.34876 4.12788 2.46657 3.66E−03phosphate 5-kinase, type I, beta (PIP5K1B), mRNA. /PROD =phosphatidylinositol-4- phosphate 5-kinase, typel, beta /FL =gb:NM_003558.1 Homo sapiens mRNA; cDNA 1.36443 4.34866 2.94283 2.92E−03DKFZp434E082 (from clone DKFZp434E082) Homo sapiens dystrophin (muscular1.40427 4.41881 3.04441 1.83E−02 dystrophy, Duchenne and Becker types),includes DXS142, DXS164, DXS206, DXS230, DXS239, DXS268, DXS269, DXS270,DXS272 (DMD), transcript variant Dp427p2, mRNA. /PROD = dystrophinDp427p2 isoform /FL = gb:NM_004010.1 Homo sapiens, clone MGC:14801,2.03928 3.96694 1.51059 5.65E−04 mRNA, complete cds. /PROD = Unknown(protein for MGC: 14801) /FL = gb:BC005997.1 Homo sapiens hypotheticalprotein −0.290244 2.2418 0.40157 6.04E−03 BC017868 (LOC159091), mRNA./FL = gb:BC017868.1 gb:NM_138819.1 ESTs, Highly similar to AF229172 1−0.475471 1.94826 0.002417 5.28E−03 class III myosin (H. sapiens) Homosapiens solute carrier family 9 0.047487 2.80601 1.19645 2.80E−03(sodiumhydrogen exchanger), isoform 5 (SLC9A5), mRNA. /PROD = solutecarrier family 9 (sodiumhydrogenexchanger), isoform 5 /FL =gb:AF111173.1 gb:NM_004594.1 Homo sapiens F37Esophageal cancer-−0.128814 3.17069 2.10422 1.54E−02 related gene-coding leucine-zippermotif (FEZ1), mRNA. /PROD = F37Esophageal cancer-relatedgene-codingleucine- zipper motif /FL = gb:AF123659.1 gb:NM_021020.1 Homosapiens deiodinase, −0.136937 5.08366 4.22897 1.41E−02 iodothyronine,type III (DIO3), mRNA. /PROD = thyroxine deiodinase type III /FL =gb:NM_001362.1 gb:S79854.1 Homo sapiens insulin-like growth factor2.6126 5.13945 3.30402 9.72E−04 binding protein 6 (IGFBP6), mRNA. /PROD= insulin-like growth factor binding protein 6 /FL = gb:BC005007.1gb:M62402.1 gb:BC003507.1 gb:NM_002178.1 Homo sapiens U2 small nuclear−0.821533 1.81533 0.096512 4.72E−02 ribonucleoprotein auxiliary factor(65 kD) (U2AF65), mRNA. /PROD = U2 small nuclear ribonucleoproteinauxiliaryfactor (65 kD) /FL = gb:NM_007279.1 Homo sapiens hypotheticalprotein −1.11464 1.81375 0.394686 9.09E−03 DKFZp434F0318(DKFZP434F0318), mRNA. /PROD = hypothetical protein DKFZp434F0318 /FL =gb:NM_030817.1 Homo sapiens BACE mRNA for beta- 0.48553 3.13284 1.445884.99E−04 site APP cleaving enzyme I-476, complete cds. /PROD = beta-siteAPP cleaving enzyme I-476 /FL = gb:AB050436.1 ESTs, Weakly similar toALUC_HUMAN 1.07951 4.06134 2.7193 2.11E−02 !!!! ALU CLASS C WARNINGENTRY !! (H. sapiens) Homo sapiens mRNA for KIAA0876 −0.432037 1.977360.073246 3.37E−02 protein, partial cds. /PROD = KIAA0876 protein Homosapiens mRNA for protein- 0.160986 2.75713 1.04172 2.11E−02 tyrosinekinase, complete cds. PROD = protein-tyrosine kinase /FL = gb:U05682.1gb:D17517.1 gb:D50479.1 KIAA0418 gene product −0.677643 1.83134 0.0343851.45E−04 Homo sapiens phosphofructokinase, 2.039 4.51747 2.703881.28E−03 liver (PFKL), mRNA. /PROD = phosphofructokinase, liver /FL =gb:NM_002626.1 gb:BC004920.1 gb:X15573.1 Homo sapiens enolase 2, (gamma,2.60624 5.37458 3.92621 3.17E−03 neuronal) (ENO2), mRNA. /PROD = enolase2, (gamma, neuronal) /FL = gb:NM_001975.1 gb:BC002745.1 gb:M22349.1 Homosapiens AD036 mRNA, complete −0.503334 2.65787 1.60278 3.61E−02 cds./PROD = AD036 /FL = gb:AF260333.1 ESTs −2.07933 1.00157 −0.1307 2.39E−02Homo sapiens keratin 19 (KRT19), 3.80582 6.96535 5.91937 2.11E−02 mRNA./PROD = keratin 19 /FL = gb:NM_002276.1 gb:BC002539.1 pleiomorphicadenoma gene-like 1 −0.646793 1.88692 0.221481 4.21E−03 /FL =gb:U72621.3 Homo sapiens, Similar to lipase protein, −1.16073 1.99940.961706 1.89E−02 clone MGC:2843, mRNA, complete cds. /PROD = Similar tolipase protein /FL = gb:NM_020676.1 gb:BC001698.1 Cluster Incl.AB002344:Human mRNA 2.15679 4.91332 3.47797 7.39E−03 for KIAA0346 gene,partial cds /cds = (0,4852) /gb = AB002344 /gi = 2280479 /ug = Hs.103915/len = 6121 Cluster Incl. N80935:zb07g06.s1 Homo 2.12419 4.48236 2.665551.50E−02 sapiens cDNA, 3 end /clone = IMAGE- 301402 /clone_end = 3 /gb =N80935 /gi = 1243636 /ug = Hs.22483 /len = 527 tryptase, alpha 2.991085.46095 3.76442 1.31E−03 ESTs, Weakly similar to Z132_HUMAN 1.048653.65906 2.12166 2.43E−02 ZINC FINGER PROTEIN 13 (H. sapiens) Homosapiens core histone 1.67293 4.73906 3.66212 3.62E−02 acroH2A2.2(MACROH2A2), mRNA. /PROD = core histone macroH2A2.2 /FL = gb:AF151534.1gb:NM_018649.1 ESTs −0.146413 2.69334 1.39451 1.02E−02 Human rho GDImRNA, complete cds. 0.019358 1.75075 −0.65155 1.85E−02 /PROD = human rhoGDI /FL = gb:M97579.1 gb:D13989.1 gb:NM_004309.1 heat shock 90 kDprotein 1, alpha 1.69225 4.20576 2.60887 2.10E−02 Homo sapiens BTB (POZ)domain 1.10085 3.7628 2.32464 1.85E−02 containing 2 (BTBD2), mRNA. /PROD= BTB (POZ) domain containing 2 /FL = gb:NM_017797.1 Homo sapiensmandaselin long form −0.148053 2.58361 1.2296 1.57E−02 mRNA, completecds. /PROD = mandaselin long form /FL = gb:AY048775.1 signal transducerand activator of −0.837261 1.90254 0.560225 8.83E−04 transcription 3(acute-phase response factor) Homo sapiens lymphocyte antigen 6 0.8663644.91832 4.92642 2.78E−02 complex, locus E (LY6E), mRNA. /PROD =lymphocyte antigen 6 complex, locus E /FL = gb:U42376.1 gb:NM_002346.1gb:U56145.1 ESTs −1.24632 1.09042 −0.61316 4.05E−02 Homo sapiens, cloneIMAGE:4047715, −2.50816 1.81 1.52693 2.63E−02 mRNA. Cluster Incl.AB002344:Human mRNA 1.89578 4.61333 3.31717 1.12E−02 for KIAA0346 gene,partial cds /cds = (0,4852) /gb = AB002344 /gi = 2280479 /ug = Hs.103915/len = 6121 Homo sapiens singed (Drosophila)-like 4.23317 6.747745.25403 5.12E−03 (sea urchin fascin homolog like) (SNL), mRNA. /PROD =singed (Drosophila)-like (sea urchin fascinhomolog like) /FL =gb:BC000521.1 gb:NM_003088.1 gb:U03057.1 gb:U09873.1 Homo sapiens mRNA;cDNA −2.72593 1.58998 1.27994 4.54E−02 DKFZp586L0120 (from cloneDKFZp586L0120). Homo sapiens mitogen-activated protein 0.647994 3.464592.28644 6.65E−03 kinase 10 (MAPK10), mRNA. /PROD = mitogen-activatedprotein kinase 10 /FL = gb:U07620.1 gb:U34819.1 gb:U34820.1gb:NM_002753.1 Homo sapiens transmembrane tyrosine −0.006089 2.960351.93237 7.69E−03 kinase mRNA, complete cds. /PROD = tyrosine kinase /FL= gb:L08961.1 Homo sapiens mRNA; cDNA 2.83663 5.30784 3.79206 6.22E−03DKFZp762H185 (from clone DKFZp762H185) tyrosine3-monooxygenasetryptophan 5- 3.48271 6.19368 4.92556 2.11E−02monooxygenase activation protein, eta polypeptide Homo sapiens mRNA;cDNA 0.581419 3.38088 2.21428 3.89E−02 DKFZp434K0621 (from cloneDKFZp434K0621); partial cds ESTs, Weakly similar to ALUF_HUMAN −1.032471.75349 0.587598 2.11E−02 !!!! ALU CLASS F WARNING ENTRY !!! (H.sapiens) tudor repeat associator with PCTAIRE 2 2.15771 4.69127 3.274512.63E−02 Homo sapiens mRNA; cDNA 4.49405 7.38448 6.33474 1.39E−02DKFZp564K1672 (from clone DKFZp564K1672); partial cds. /PROD =hypothetical protein ESTs −0.184212 2.15367 0.554696 1.15E−02 Homosapiens bHLH factor Hes4 0.47816 3.40725 2.40001 3.16E−02 (LOC57801),mRNA. /PROD = bHLH factor Hes4 /FL = gb:NM_021170.1 gb:AB048791.1 Homosapiens guanylate cyclase −0.714216 1.95339 0.684983 3.38E−03 activator1B (retina) (GUCA1B), mRNA. /PROD = guanylate cyclase activator 1B(retina) /FL = gb:M95174.1 b:NM_002098.1 gb:M97496.1 KIAA0918 protein−1.39708 0.961939 −0.57499 1.40E−02 Homo sapiens mRNA for KIAA11610.781796 3.32846 1.98143 6.60E−03 protein, partial cds. /PROD = KIAA1161protein Homo sapiens, Similar to B9 protein, 1.43881 3.80457 2.292058.62E−05 clone MGC:11339, mRNA, complete cds. /PROD = Similar to B9protein /FL = gb:BC002944.1 hypothetical protein FLJ12666 2.340875.11201 4.02837 1.97E−02 Homo sapiens FLICE-like inhibitory 0.1357834.45631 4.92758 1.96E−02 protein short form mRNA, complete cds. /PROD =FLICE-like inhibitory protein short form /FL = gb:U97075.1 Homo sapiensolfactory receptor, family −0.309574 2.08723 0.636487 4.36E−03 52,subfamily A, member 1 (OR52A1), mRNA. /PROD = olfactory receptor, family52, subfamily A,member 1 /FL = gb:NM_012375.1 Human DNA sequence fromclone RP4- 2.82768 5.54517 4.43909 2.04E−03 781L3 on chromosome1p34.3-36.11 Contains a pseudogene similar to IFITM3 (interferoninducedntransmembrane protein 3 (1- 8U)), STSs and GSSs Homo sapiensKIAA0127 gene product 2.05236 4.43794 3.00216 3.12E−02 (KIAA0127), mRNA./PROD = KIAA0127 gene product /FL = gb:D50917.1 gb:NM_014755.1 Homosapiens clone HB-2 mRNA −0.503949 2.27896 1.25171 3.01E−02 sequencehypothetical protein FLJ23091 −0.76092 3.70916 3.04154 2.45E−02 ESTs−0.403665 2.01868 0.643615 5.11E−04 ESTs 2.14401 4.70353 3.471143.15E−03 Homo sapiens regulator of G protein 3.0091 5.37511 3.951321.29E−03 signalling 5 (RGS5) mRNA, complete cds. /PROD = regulator of Gprotein signalling 5 /FL = gb:AF493929.1 endothelin receptor type A−1.83423 2.54948 1.95432 1.51E−02 /FL = gb:NM_001957.1 gb:L06622.1 H.sapiens skeletal embryonic myosin 0.690058 3.3896 2.36018 3.27E−02 lightchain 1 (MLC1) mRNA. /PROD = myosin light chain 1 Homo sapiensKruppel-like factor 8 0.326119 2.825 1.63929 3.92E−02 (KLF8), mRNA./PROD = Kruppel-like factor 8 /FL = gb:U28282.1 gb:NM_007250.1hypothetical protein DKFZp434F2322 0.070576 2.58185 1.42121 5.81E−03Homo sapiens transcription factor 2, −1.12537 3.45355 2.54347 1.15E−02hepatic; LF-B3; variant hepatic nuclear factor (TCF2), transcriptvariant a, mRNA. /PROD = transcription factor 2, isoform a /FL =gb:NM_000458.1 H2A histone family, member X 1.37085 2.48515 −0.040571.34E−02 Homo sapiens H1 histone family, 3.21701 5.68105 4.509694.29E−03 member X (H1FX), mRNA. /PROD = H1 histone family, member X /FL= gb:D64142.1 gb:BC000426.1 gb:NM_006026.1 ESTs, Weakly similar toKIAA1399 −0.831241 2.82364 2.79997 4.18E−02 protein (H. sapiens) Homosapiens PNAS-145 mRNA, 2.84962 5.38084 4.2868 8.61E−03 complete cds./PROD = PNAS-145 /FL = gb:U03105.1 gb:NM_006813.1 gb:AF279899.1 Homosapiens erythrocyte membrane −0.306603 2.11391 0.923806 4.81E−03 proteinband 4.9 (dematin) (EPB49), mRNA. /PROD = erythrocyte membrane proteinband 4.9 (dematin) /FL = gb:NM_001978.1 gb:U28389.1 ESTs 0.7835453.23146 2.07738 2.13E−02 ESTs 1.17515 3.61815 2.46265 1.56E−03 nudix(nucleoside diphosphate linked 2.09472 5.82355 5.68859 3.63E−02 moietyX)-type motif 4 Novel human mRNA from chromosome −0.533665 3.446793.05803 3.99E−02 22. /PROD = hypothetical protein Homo sapiens partialRNA for putative 1.55224 3.89366 2.67866 1.53E−03 nuclear factor. /PROD= putative nuclear factor /FL = gb:NM_017688.1 Homo sapiensBCL2adenovirus E1B 4.51142 6.99506 5.92755 2.25E−03 19 kD-interactingprotein 3 (BNIP3), nuclear gene encoding mitochondrial protein, mRNA./PROD = BCL2adenovirus E1B 19 kD- interacting protein 3 /FL =gb:AF002697.1 gb:NM_004052.2 gb:U15174.1 Homo sapiens bone morphogenetic−0.6099 1.76351 0.614791 6.46E−03 protein 5 (BMP5), mRNA. /PROD = bonemorphogenetic protein 5 /FL = gb:M60314.1 gb:NM_021073.1 Homo sapiensnuclear receptor −0.213822 4.14059 3.29782 3.42E−02 subfamily 0, groupB, member 1 (NROB1), mRNA. /PROD = adrenal hypoplasia protein /FL =gb:NM_000475.2 Homo sapiens mRNA; cDNA 1.76846 4.196 3.11284 1.23E−02DKFZp434P228 (from clone DKFZp434P228) Homo sapiens pilin-liketranscription 3.14467 5.51066 4.38212 3.80E−03 factor (PILB), mRNA./PROD = pilin-like transcription factor /FL = gb:AF122004.1gb:NM_012228.1 Homo sapiens complement component −1.89639 1.656371.59643 1.74E−02 5 (C5), mRNA. /PROD = complement component 5 /FL =gb:M57729.1 gb:NM_001735.1 Homo sapiens adaptor-related protein−0.208283 2.17404 1.07398 1.39E−02 complex 3, beta 2 subunit (AP3B2),mRNA. /PROD = adaptor-related protein complex 3, beta 2subunit /FL =gb:AF022152.1 gb:NM_004644.1 gb:U37673.1 Homo sapiens dynein, axonemal,light 0.458271 2.88755 1.8494 3.66E−03 polypeptide 4 (DNAL4), mRNA./PROD = dynein, axonemal, light polypeptide 4 /FL = gb:BC002968.1gb:NM_005740.1 Homo sapiens, Similar to RIKEN cDNA 0.087468 2.458671.42701 3.35E−03 C330013D18 gene, clone MGC:11226, mRNA, complete cdsHomo sapiens cDNA: FLJ22731 fis, −2.30948 1.62817 2.24143 4.15E−02 cloneHSI15841. ESTs 0.532996 4.6172 3.79228 2.76E−02 Homo sapiens CXCR4 geneencoding 3.11086 7.24917 6.36084 3.13E−02 receptor CXCR4 stanniocalcin 12.48686 6.20245 5.70081 4.94E−02 KIAA0761 protein −2.09591 1.579351.05907 1.86E−02 ESTs −1.16374 2.24964 2.52108 3.83E−02 Homo sapienscDNA FLJ36116 fis, 3.02587 6.7146 6.15456 3.88E−02 clone TESTI2022338.Homo sapiens, clone MGC:24252 −1.53346 2.53854 1.59251 3.91E−03IMAGE:3932604, mRNA, complete cds. /PROD = Unknown (protein forMGC:24252) /FL = gb:BC014364.1 Homo sapiens cDNA FLJ13392 fis, −1.901841.76129 1.16856 1.47E−02 clone PLACE1001280 Homo sapiens hypotheticalprotein 1.70617 5.11419 4.73391 2.71E−02 FLJ12538 similar to ras-relatedprotein RAB17 (FLJ12538), mRNA. /PROD = hypothetical protein FLJ12538similar toras-related protein RAB17 /FL = gb:AL136645.1 gb:NM_022449.1Human DNA sequence from clone −0.889422 2.90076 2.11955 3.52E−02RP11-446H13 on chromosome 10. Contains the 3 end of the gene for a novelprotein similar to KIAA1059 (ortholog of mouse VPS10 domain receptorprotein SORCS), an RPL23A (60S ribosmal protein 23A) pseudogene, ESTs,STSs an Homo sapiens calmegin (CLGN), −0.104278 2.97817 3.11092 1.89E−02mRNA. /PROD = calmegin /FL = gb:NM_004362.1 gb:D86322.1 Homo sapienstestican 3 (HSAJ1454), −2.81226 0.7939 1.55068 3.99E−02 mRNA. /PROD =testican 3 /FL = gb:NM_016950.1 gb:BC000460.1 gb:BC003017.1 Homo sapiensgelsolin (amyloidosis, 1.6198 4.54047 4.45008 1.07E−02 Finnish type)(GSN), mRNA. /PROD = gelsolin (amyloidosis, Finnish type) /FL =gb:NM_000177.1 Homo sapiens endothelin receptor type 0.763007 4.059833.55203 3.99E−02 A (EDNRA), mRNA. /PROD = endothelin receptor type A /FL= gb:NM_001957.1 gb:L06622.1 Homo sapiens cDNA: FLJ22808 fis, −0.3674492.7584 2.41394 4.31E−02 clone KAIA2925. Homo sapiens hypotheticalprotein 1.59498 4.6335 4.89157 4.85E−02 FLJ10312 (FLJ10312), mRNA. /PROD= hypothetical protein FLJ10312 /FL = gb:NM_030672.1 Homo sapiens lipasemRNA, complete −2.2131 1.0208 0.55849 3.57E−02 cds. /PROD = lipase /FL =gb:AF225418.1 Homo sapiens clone FLC1492 PRO3121 −0.300037 −0.46894−3.39203 1.80E−02 mRNA, complete cds. /PROD = PRO3121 /FL =gb:AF130082.1 protease, serine, 4 (trypsin 4, brain) −0.345569 2.862563.35886 4.65E−02 Homo sapiens ret finger protein-like 2 −2.15511 1.17970.503917 1.94E−02 (RFPL2), mRNA. /PROD = ret finger protein-like 2 /FL =gb:NM_006605.1 Homo sapiens beta3GaINAcT-1 mRNA −2.73257 0.21269 −0.09842.48E−02 for globoside synthase, complete cds, clone:type 2. /PROD =globoside synthase /FL = gb:AB050856.1 Homo sapiens hypothetical protein−3.34565 −0.42738 −0.71255 3.07E−02 FLJ11155 (FLJ11155), mRNA. /PROD =hypothetical protein FLJ11155 /FL = gb:NM_018342.1 Homo sapiens, dualspecificity 2.11487 5.16681 5.60057 3.11E−02 phosphatase 4, cloneMGC:3713, mRNA, complete cds. /PROD = dual pecificity phosphata e 4 /FL= gb:NM_001394.2 gb:BC002671.1 gb:U48807.1 gb:U21108.1 Homo sapiens MAD(mothers against 0.917516 4.29847 3.5309 2.44E−02 decapentaplegic,Drosophila) homolog 6 (MADH6), mRNA. /PROD = MAD (mothers againstdecapentaplegic, Drosophila) homolog 6 /FL = gb:U59914.1 gb:NM_005585.1secreted frizzled-related protein 1 1.64933 4.41286 4.21799 4.12E−02 /FL= gb:AF056087.1 gb:NM_003012.2 gb:AF017987.1 gb:AF001900.1 Homo sapienssecreted apoptosis 3.18446 5.9316 5.74251 3.31E−02 related protein 2(SARP2) mRNA, complete cds. /PROD = secreted apoptosis related protein 2/FL = gb:AF056087.1 gb:NM_003012.2 gb:AF017987.1 gb:AF001900.1 KIAA0882protein 2.05587 4.63577 4.67451 4.89E−02 Homo sapiens nudix (nucleoside3.91519 6.54632 6.42767 4.63E−02 diphosphate linked moiety X)-type motif4 (NUDT4), mRNA. /PROD = nudix (nucleoside diphosphate linkedmoietyX)-type motif 4 /FL = gb:NM_019094.1 gb:AF191653.1 gb:AF191649.1gb:AF191650.1 Homo sapiens cDNA FLJ31061 fis, −1.30452 2.06218 1.207491.72E−02 clone HSYRA2000927. Homo sapiens diphosphoinositol 2.626125.53515 5.12935 3.96E−02 polyphosphate phosphohydrolase type 2 beta(NUDT4) mRNA, complete cds. /PROD = diphosphoinositol polyphosphatephosphohydrolasetype 2 beta /FL = gb:NM_019094.1 gb:AF191653.1gb:AF191649.1 gb:AF191650.1 Homo sapiens titin (TTN), mRNA. −0.4661542.46856 2.00309 4.18E−03 /PROD = titin /FL = gb:NM_003319.1arboxypeptidase E 2.72316 5.80072 5.15668 2.99E−02 /FL = gb:NM_001873.1ESTs −1.80443 0.692933 0.613229 1.46E−03 Homo sapiens hypotheticalprotein −1.60435 1.6149 2.4204 2.10E−02 FLJ39502 (FLJ39502), mRNA. /FL =gb:NM_173648.1 Homo sapiens renal tumor antigen −0.305406 2.4556 2.091363.30E−02 (RAGE), mRNA. /PROD = renal tumor antigen /FL = gb:NM_014226.1gb:AB022694.1 T-box 3 (ulnar mammary syndrome) −1.70952 1.31576 0.6795841.29E−02 Homo sapiens mitogen-activated protein −1.14241 1.82177 1.239282.48E−02 kinase kinase kinase 8 (MAP3K8), mRNA. /PROD =mitogen-activated protein kinase kinase kinase8 /FL = gb:NM_005204.1gb:D14497.1 Homo sapiens enoyl-Coenzyme A, 0.226733 3.18231 2.599386.22E−03 hydratase3-hydroxyacyl Coenzyme A dehydrogenase (EHHADH),nuclear gene encoding mitochondrial protein, mRNA. /PROD =enoyl-Coenzyme A, hydratase3-hydroxyacylCoenzyme A dehydrogenase /FL =gb:NM_001966.1 gb:L07077.1 ESTs 1.65525 3.9984 3.98375 1.97E−02 Homosapiens secreted frizzled-related 3.91678 7.18429 6.24313 2.35E−02protein 1 (SFRP1), mRNA. /PROD = secreted frizzled-related protein 1 /FL= gb:AF056087.1 gb:NM_003012.2 gb:AF017987.1 gb:AF001900.1 ESTs 2.286595.21675 4.59884 3.11E−02 Homo sapiens selenoprotein P, plasma, 2.733325.13625 5.04138 1.75E−02 1 (SEPP1), mRNA. /PROD = selenoprotein Pprecursor /FL = gb:NM_005410.1 hypothetical protein FLJ12838 2.215225.4785 4.50835 4.55E−02 /FL = gb:NM_024641.1 Homo sapiens mRNA; cDNA−2.59761 0.092924 −0.31569 4.19E−02 DKFZp761M1216 (from cloneDKFZp761M1216) Homo sapiens KPL1 (KPL1) mRNA, −0.08886 2.51294 2.159671.97E−02 complete cds. /PROD = KPL1 /FL = gb:AF081583.1 gb:U89715.1gb:NM_021200.1 Homo sapiens, synovial sarcoma, X −1.57209 1.398692.12662 6.84E−03 breakpoint 1, clone MGC:5162, mRNA, complete cds. /PROD= synovial sarcoma, X breakpoint 1 /FL = gb:BC001003.2 gb:NM_005635.1Homo sapiens, Similar to testican 3, −2.778 0.563149 1.66935 1.87E−02clone MGC:8506, mRNA, complete cds. /PROD = Similar to testican 3 /FL =gb:NM_016950.1 gb:BC000460.1 gb:BC003017.1 ESTs 0.502949 3.0682 2.728742.02E−02 Homo sapiens cDNA FLJ32963 fis, 1.49644 3.87336 3.684786.89E−03 clone TESTI2008405. triadin /FL = gb:U18985.1 −1.61408 0.7130830.566175 1.87E−02 gb:NM_006073.1 Homo sapiens adlican mRNA, complete0.872786 3.20404 3.02406 4.21E−02 cds. /PROD = adlican /FL =gb:AF245505.1 Human midkine mRNA, complete cds. 3.55275 5.93819 5.683631.89E−02 /PROD = midkine /FL = gb:NM_002391.1 gb:M69148.1 Homo sapiens,synovial sarcoma, X −0.806785 1.90972 2.5215 4.17E−02 breakpoint 4,clone MGC:12411, mRNA, complete cds. /PROD = synovial sarcoma, Xbreakpoint 4 /FL = gb:BC005325.1 ESTs −3.61193 −0.9408 −1.53439 2.43E−03ESTs −0.619714 1.93617 1.44843 4.14E−02 ESTs 0.446983 3.14315 2.486761.98E−02 Homo sapiens flavin containing −0.476203 1.99948 1.554913.73E−02 monooxygenase 5 (FMO5), mRNA. /PROD = flavin containingonooxygenase 5 /FL = gb:L37080.1 gb:NM_001461.1 Homo sapiensaminopeptidase A −0.758987 2.07596 1.20831 3.00E−02 mRNA, complete cds./PROD = aminopeptidase A /FL = gb:L12468.1 gb:NM_001977.1 gb:L14721.1Homo sapiens hypothetical protein −0.713164 2.03145 1.24823 2.17E−03DKFZp761H1710 (DKFZP761H1710), mRNA. /PROD = hypothetical proteinDKFZp761H1710 /FL = gb:NM_031297.1 ESTs −1.94194 0.608912 0.0132381.72E−02 Homo sapiens sialyltransferase 8 (alpha- −1.07243 1.838890.855205 2.16E−02 2, 8-polysialytransferase) D (SIAT8D), mRNA. /PROD =sialyltransferase 8 (alpha-2,8-polysialytransferase) D /FL =gb:NM_005668.1 gb:L41680.1 Homo sapiens, clone IMAGE:5194204, −2.132560.383746 1.02639 4.55E−02 mRNA. hypothetical protein MGC4342 −0.4030182.01722 1.41263 1.97E−02 /FL = gb:NM_024329.1 gb:BC003033.1 Homo sapiensPHD finger protein 1 0.620171 2.95191 2.42296 2.32E−02 (PHF1),transcript variant 2, mRNA. /PROD = PHD finger protein 1, isoform b /FL= gb:NM_024165.1 gb:AF052205.1 0.436893 3.11051 2.23625 5.44E−03actinin, alpha 4 Homo sapiens PCTAIRE protein kinase 2.38171 4.870714.16957 2.90E−03 1 (PCTK1), mRNA. /PROD = PCTAIRE protein kinase 1 /FL =gb:NM_006201.1 Human DNA sequence from clone 0.167347 2.56439 1.937363.93E−02 RP11-165F24 on chromosome 9. Contains the 3 end of the gene fora novel protein (similar to Drosophila CG6630 and CG11376, KIAA1058, ratTRG), an RPL12 (60S ribosomal protein L12) pseudogene, ESTs, STSs, GSSsand a C. . . insulin-like growth factor binding protein 0.921068 3.602792.67905 4.80E−02 3 /FL = gb:NM_000598.1 Homo sapiens, cloneIMAGE:3840937, 3.59778 6.12936 6.92649 8.62E−05 mRNA, partial cds. /PROD= Unknown (protein for IMAGE:3840937) Homo sapiens phosphoglucomutase 14.74364 7.09524 6.43056 2.21E−02 (PGM1), mRNA. /PROD =phosphoglucomutase 1 /FL = gb:NM_002633.1 gb:BC001756.1 gb:M83088.1apolipoprotein C-I 2.77701 5.23054 4.44049 3.39E−02 Homo sapiens insulininduced gene 1 2.11462 4.44128 3.77765 3.21E−02 (INSIG1), mRNA. /PROD =insulin induced gene 1 /FL = gb:NM_005542.1 Human a6(IV) collagen(COL4A6) 0.798849 3.23623 2.41372 1.38E−03 mRNA, complete cds. /PROD = Atype IV collagen /FL = gb:U04845.1 Homo sapiens mRNA for alpha 1,6-1.31479 3.6699 2.90742 3.48E−02 fucosyltransferase, complete cds. /PROD= alpha 1,6-fucosyltransferase /FL = gb:AB049740.2 ESTs, Moderatelysimilar to Six5 0.434259 2.81512 1.9556 1.23E−02 (M. musculus) solutecarrier family 2 (facilitated glucose 5.23156 7.63734 6.74444 1.99E−02transporter), member 3 /FL = gb:NM_006931.1 gb:M20681.1 Human acidsphingomyelinase (ASM) 1.03965 3.45689 2.52125 2.62E−02 mRNA, completecds. /PROD = acid sphingomyelinase /FL = gb:NM_000543.1 gb:M59916.1ESTs, Weakly similar to unnamed 3.0131 5.41021 4.46614 4.79E−03 proteinproduct (H. sapiens) Homo sapiens cDNA FLJ33178 fis, 0.137476 −1.9718−4.73502 2.23E−03 clone ADRGL2002753. Homo sapiens, parathyroidhormone-like 2.46289 −0.34647 −1.96791 3.05E−02 hormone, cloneMGC:14611, mRNA, complete cds. /PROD = parathyroid hormone-like hormone/FL = gb:BC005961.1 Homo sapiens colon carcino a related 2.50614−0.01889 −1.04926 2.52E−02 protein (LOC51159), mRNA. /PROD = coloncarcinoma related protein /FL = gb:NM_016206.1 gb:AF099505.1 KIAA0036gene product 3.05174 −0.66523 −2.2068 2.62E−02 Homo sapiens cDNAFLJ13536 fis, 4.27968 1.86625 1.70965 1.11E−02 clone PLACE1006521 Homosapiens cDNA: FLJ22463 fis, 3.68063 1.24806 1.39728 1.42E−02 cloneHRC10126 Homo sapiens UDP-N- 2.71164 0.270906 0.433883 1.98E−02acetylglucosamine:a-1,3-D-mannoside beta-1,4-N-acetylglucosaminyltransferase IV- homolog (HGNT-IV-H), mRNA. /PROD =UDP-N-acetylglucosamine:a- 1,3-D-mannosidebeta-1,4-N-acetylglucosaminyltransferase IV- homolog /FL = gb:AB024729.1 gb:NM_0tissue factor pathway inhibitor 2 4.19529 1.63281 1.58393 7.53E−03 /FL =gb:D29992.1 gb:L27624.1 gb:NM_006528.1 gb:BC005330.1 ESTs 2.89240.521234 0.762594 2.82E−02 putative gene product −0.518738 −0.666151.82038 4.91E−02 Homo sapiens mRNA for KIAA1758 2.42082 −0.039230.213473 1.25E−02 protein, partial cds. /PROD = KIAA1758 proteinprostaglandin E receptor 4 (subtype 2.58497 0.196846 0.527172 1.42E−02EP4) /FL = gb:L25124.1 gb:D28472.1 gb:NM_000958.1 gb:L28175.1 Homosapiens cDNA: FLJ22463 fis, 2.85068 0.473875 0.830447 1.97E−02 cloneHRC10126 Homo sapiens heparan sulfate 3.18462 0.571052 0.709206 4.54E−02(glucosamine) 3-O-sulfotransferase 2 (HS3ST2), mRNA. /PROD = heparansulfate D-glucosaminyl3-O- sulfotransferase 2 /FL = gb:AF105375.1gb:AF105374.1 gb:NM_006043.1 Homo sapiens schwannomin interacting 5.59573.21054 3.57915 2.11E−02 rotein 1 (SCHIP-1), mRNA. /PROD = schwannomininteracting protein 1 /FL = gb:AF145713.1 gb:NM_014575.1 2.11886−0.24936 0.138069 2.55E−03 Human lysyl oxidase (LOX) gene, exon 7 Humannephropontin mRNA, complete 7.82555 5.27856 5.06542 4.61E−02 cds. /PROD= nephropontin /FL = gb:M83248.1 Human mRNA for KIAA0386 gene, 3.92131.56305 1.13663 1.93E−02 complete cds. /FL = gb:AB002384.1 ESTs 3.221460.851355 0.404487 3.41E−03 Human glioma pathogenesis-related 3.201870.502872 0.348681 1.34E−02 protein (GliPR) mRNA, complete cds. /PROD =glioma pathogenesis-related protein /FL = gb:NM_006851.1 gb:U16307.1ESTs 2.96697 0.440894 0.773495 1.97E−02 Homo sapiens cDNA FLJ13384 fis,5.9221 3.48419 3.9531 3.38E−02 clone PLACE1001062, highly similar toHomo sapiens mRNA for lysine- ketoglutarate reductasesaccharopinedehydrogenase. Homo sapiens mRNA; cDNA 6.06894 3.31919 3.4798 4.97E−02DKFZp76111912 (from clone DKFZp76111912) ESTs 4.12419 1.53997 1.882395.68E−03 Homo sapiens fibroblast growth factor 2 5.49297 2.64534 2.760544.71E−02 (basic) (FGF2), mRNA. /PROD = fibroblast growth factor 2(basic) /FL = gb:NM_002006.1 gb:M27968.1 ribosomal protein L34pseudogene 1 3.45116 2.78236 5.10812 2.42E−02 HIV-1 rev binding protein2 4.4245 1.47599 1.52824 2.10E−02 H. sapiens FGF gene, exon 3 2.04849−0.34703 −0.95745 3.33E−02 /FL = gb:NM_000800.1 gb:M13361.1 ESTs 2.03304−0.9164 −0.83676 4.19E−02 ESTs 2.87393 0.428858 1.02011 9.72E−04 Homosapiens clone 23700 mRNA 4.60148 1.64243 1.72052 4.74E−02 sequence STs,Highly similar to S21424 nestin 3.08579 0.725853 1.40345 1.99E−02 (H.sapiens) Homo sapiens actin, gamma 2, smooth 7.17155 4.15208 4.185514.72E−02 muscle, enteric (ACTG2), mRNA. /PROD = actin, gamma 2propeptide /FL = gb:NM_001615.2 hypothetical protein FLJ22833 2.807850.480636 1.25154 2.43E−02 Homo sapiens LIM homeobox protein 6 2.52571−0.06036 0.459727 4.37E−02 (LHX6), mRNA. /PROD = LIM homeobox protein 6/FL = gb:AB031041.1 gb:NM_014368.1 gb:AL136570.1 Homo sapiens, cloneIMAGE:5271039, 3.05576 0.189935 0.452681 4.05E−02 mRNA. Homo sapienscAMP response element- 2.90971 0.487264 1.21334 4.24E−02 binding proteinCRE-BPa (H_GS165L15.1), mRNA. /PROD = CAMP response element-bindingprotein CRE- BPa /FL = gb:NM_004904.1 gb:L05911.1 G protein-coupledreceptor 1 1.15804 −1.24345 −0.49352 2.28E−03 /FL = gb:NM_005279.1 Homosapiens neuropilin (NRP) and 3.25525 0.20233 0.301354 2.83E−02 tolloid(TLL)-like 1 (NETO1), transcript variant 3, mRNA. /PROD = neuropilin-and tolloid-like protein 1 isoform 3precursor /FL = gb:AF448838.1gb:NM_138966.2 Homo sapiens mRNA for KIAA0559 2.37088 −0.29358 0.1990282.81E−02 protein, partial cds. /PROD = KIAA0559 protein Human65-kilodalton phosphoprotein 3.25522 0.620942 1.15317 2.88E−02 (p65)mRNA, complete cds. /PROD = phosphoprotein p65 /FL = gb:M22300.1gb:NM_002298.2 gb:J02923.1 Human DNA sequence from clone 4.77522 2.189712.77459 4.04E−02 RP11-31K16 on chromosome 9. Contains a snoRNA bindingdomain pseudogene, the ELAVL2 gene for LAV (embryonic lethal, abnormalGSSs and a CpG island vision, Drosophila)-like 2, ESTs, STSs, GSSs and aCpG island hypothetical protein FLJ11006 1.75389 −0.66646 0.1005917.78E−04 Homo sapiens mRNA; cDNA 3.97344 1.32598 1.8683 3.19E−02DKFZp566A1046 (from clone DKFZp566A1046) ESTs 4.13356 1.60769 2.274151.85E−02 H. sapiens gene from PAC 106H8. 3.05906 −0.08711 −0.138454.34E−02 Human T-cell receptor rearranged beta- 3.43862 0.94127 1.645145.31E−03 chain V-region (V-D-J) mRNA, complete cds. /FL = gb:M15564.1peptidylprolyl isomerase A (cyclophilin 1.76352 0.908832 3.261242.19E−02 A) Homo sapiens mRNA for KIAA1597 6.20675 3.06104 3.124031.78E−02 protein, partial cds. /PROD = KIAA1597 protein HIV-1 revbinding protein 2 3.54001 0.617783 0.325316 3.52E−02 Homo sapiensnidogen 2 (NID2), mRNA. 3.39549 0.915898 1.65422 4.70E−03 /PROD =nidogen 2 /FL = gb:NM_007361.1 gb:D86425.1 Homo sapiens mRNA; cDNA3.49826 0.859233 1.46022 3.01E−02 DKFZp566A1046 (from cloneDKFZp566A1046) Homo sapiens muscleblind (Drosophila)- 3.53962 0.5081660.298438 3.90E−02 like (MBNL), mRNA. /PROD = muscleblind(Drosophila)-like /FL = gb:NM_021038.1 gb:AB007888.1 hypotheticalprotein FLJ20163 5.26708 2.17148 2.01643 3.33E−02 KIAA0455 gene product3.25374 0.057742 −0.01612 2.66E−02 hypothetical protein FLJ22833 2.932290.533105 1.40606 3.19E−02 /FL = gb:NM_022837.1 ESTs 2.19313 −0.328220.427128 1.87E−02 Homo sapiens NADPH oxidase 4 5.57794 2.61602 2.284624.04E−02 (NOX4), mRNA. /PROD = NADPH oxidase 4 /FL = gb:AF261943.1gb:NM_016931.1 gb:AF254621.1 gb:AB041035.1 STs 3.3266 0.840887 1.696050.00 +00 ESTs 1.99635 −0.56359 0.236236 1.39E−02 Homo sapiensserumglucocorticoid 4.81433 2.46478 3.47797 2.02E−02 regulated kinase(SGK), mRNA. /PROD = serumglucocorticoid regulated kinase /FL =gb:BC001263.1 gb:NM_005627.1 gb:AF153609.1 A kinase (PRKA) anchorprotein 2 6.03149 3.65135 4.63469 1.17E−02 ESTs 3.8522 1.17894 0.4833673.08E−02 Homo sapiens similar to rat 1.74077 −1.45032 −1.24979 5.69E−04tricarboxylate carrier-like protein (BA108L7.2), mRNA. /PROD = similarto rat tricarboxylate carrier-likeprotein /FL = gb:NM_030971.1 Homosapiens, lectin, galactoside- 4.53668 1.85893 2.59596 3.84E−03 binding,soluble, 3 (galectin 3), clone MGC:2058, mRNA, complete cds. /PROD =lectin, galactoside-binding, soluble, 3(galectin 3) /FL = gb:NM_002306.1gb:M35368.1 gb:BC001120.1 gb:M36682.1 gb:M57710.1 gb:AB006780.1 ESTs1.06738 −1.51321 −0.67814 7.68E−03 Homo sapiens heptacellular carcinoma5.55437 3.02649 3.91576 2.22E−02 novel gene-3 protein (LOC51339), mRNA./PROD = heptacellular carcinoma novel gene-3 protein /FL =gb:NM_016651.2 gb:AF251079.2 Homo sapiens, Similar to transforming4.82642 2.07249 2.74019 1.03E−02 growth factor beta 1 induced transcript1, clone MGC:4078, mRNA, complete cds. /PROD = Similar to transforminggrowth factor beta 1induced transcript 1 /FL = gb:NM_015927.1gb:BC001830.1 gb:AF116343.1 ESTs, Highly similar to FXD3_HUMAN 3.815231.18624 1.98102 4.17E−02 FORKHEAD BOX PROTEIN D3 (H. sapiens) STs4.52094 1.70664 2.3196 2.78E−02 Homo sapiens potassium 3.68048 0.8973691.5433 1.32E−02 intermediatesmall conductance calcium- activatedchannel, subfamily N, member 2 (KCNN2), mRNA. /PROD = potassiumintermediatesmall conductancecalcium- activated channel, subfamily N,member 2 /FL = gb:NM_021614.1 gb:AF239613.1 Homo sapiens transportersimilar to 5.68324 3.35483 4.46553 3.00E−03 yeast MRS2 (MRS2L), mRNA./PROD = transporter similar to yeast MRS2 /FL = gb:AF288288.1gb:NM_020662.1 Homo sapiens a disintegrin-like and 4.54713 2.071521.10726 5.91E−03 metalloprotease (reprolysin type) with thrombospondintype 1 motif, 5 (aggrecanase-2) (ADAMTS5), mRNA. /PROD = a disintegrinand metalloprotease withthrombospondin motifs-5 preproprotein /FL =gb:NM_007038.1 gb:AF14209 3.85003 0.823769 0.407316 3.76E−02 HIV-1 revbinding protein 2 Human, parathyroid-like protein 1.73564 −1.5631−1.41799 4.04E−02 (associated with humoral hypercalcemia of malignancy)mRNA, complete cds. /FL = gb:J03580.1 ESTs 5.27633 2.76577 3.715421.39E−02 ESTs 1.7022 −1.29085 −0.81236 1.72E−02 pyruvate dehydrogenasephosphatase 6.62966 4.26189 5.37384 1.04E−03 /FL = gb:NM_018444.1gb:AF155661.1 3.90566 1.11378 1.80307 1.57E−02 ESTs Homo sapiens DRM(DRM) mRNA, 6.16645 3.12369 3.57831 1.59E−02 complete cds. /PROD = DRM/FL = gb:NM_013372.1 gb:AF110137.2 gb:AF045800.1 gb:AF154054.1 Homosapiens cDNA: FLJ22769 fis, 2.36483 −0.5543 0.025213 1.45E−04 cloneKAIA1316 ESTs 4.31647 1.44801 2.11226 2.19E−02 STs 1.24891 −1.42983−0.57145 1.29E−02 Homo sapiens mRNA; cDNA 2.85031 0.231102 1.164391.08E−02 DKFZp586N012 (from clone DKFZp586N012) jagged 1 (Alagillesyndrome) 2.54951 0.151382 1.31733 4.46E−03 ESTs, Weakly similar to138588 reverse 1.16209 −1.26828 −0.1187 3.40E−02 transcriptase homolog(H. sapiens) KIAA1339 protein 4.41803 2.09451 3.35786 3.90E−02 ESTs3.83407 0.892884 1.56759 8.98E−03 DKFZP434D156 protein 4.42983 2.051543.28988 1.60E−02 Homo sapiens mRNA expressed only in 3.84408 0.7050311.20856 2.25E−02 placental villi, clone SMAP41. RAS guanyl releasingprotein 2 (calcium 2.7724 0.235409 1.34452 1.96E−02 and DAG-regulated)KIAA0164 gene product 4.99867 2.5327 3.72535 2.85E−02 /FL =gb:NM_014739.1 gb:D79986.1 Homo sapiens mRNA; cDNA 5.14752 2.612173.76652 3.78E−02 DKFZp761M0111 (from clone DKFZp761M0111) Homo sapiensNPD009 mRNA, 2.15383 −0.27664 0.990295 2.09E−03 complete cds. /PROD =NPD009 /FL = gb:NM_020686.1 gb:AF237813.1 3.23823 0.467215 1.396287.60E−03 muscleblind (Drosophila)-like /FL = gb:NM_021038.1gb:AB007888.1 4.49318 1.35004 1.91395 3.79E−02 ESTs muscleblind(Drosophila)-like 1.84877 −0.57315 0.732468 8.06E−03 /FL =gb:NM_021038.1 gb:AB007888.1 6.068 3.42334 4.51189 1.89E−02 ESTs ESTs,Weakly similar to unnamed 3.24528 0.740266 1.98306 3.89E−04 proteinproduct (H. sapiens) Human complement cytolysis inhibitor 6.507664.03647 5.33533 1.77E−02 (CLI) mRNA, complete cds. /FL = gb:J02908.1gb:NM_001831.1 gb:M64722.1 gb:M25915.1 ESTs 5.36759 2.42249 3.247653.23E−03 ESTs 0.957239 −1.43055 −0.04796 5.55E−04 transporter similar toyeast MRS2 3.77395 1.18311 2.36844 1.83E−02 Homo sapiens, Similar toregulator for 5.09957 2.72179 4.14116 2.72E−02 ribosome resistancehomolog (S. cerevisiae), clone MGC:2755, mRNA, complete cds. /PROD =Similar to regulator for ribosome resistancehomolog (S. cerevisiae) /FL= gb:BC001811.1 SWISNF related, matrix associated, 2.54229 −0.292260.695255 1.89E−02 actin dependent regulator of chromatin, subfamily a,member 2 /FL = gb:NM_003070.1 gb:D26155.1 Homo sapiens mRNA; cDNA3.34446 0.795373 2.07147 1.03E−02 DKFZp586L2424 (from cloneDKFZp586L2424) ESTs 4.63117 2.08252 3.36946 2.25E−03 Homo sapiensbicarbonate transporter- 3.17407 0.745852 2.17046 1.71E−03 relatedprotein BTR1 mRNA, complete cds. /PROD = bicarbonate transporter-related protein BTR1 /FL = gb:AF336127.1 ESTs 1.08377 −1.27197 0.2283054.76E−02 ESTs 2.94657 0.127188 1.20032 1.43E−02 Homo sapiens mRNA; cDNA2.47798 −0.26439 0.894731 4.51E−03 DKFZp434B2016 (from cloneDKFZp434B2016). ESTs 2.39983 −1.04089 −0.57976 3.37E−02 Homo sapienssynaptotagmin interacting 2.11009 −0.57109 0.653181 1.78E−02 proteinSTIP1 mRNA, partial cds. /PROD = synaptotagmin interacting protein STIP1Homo sapiens connexin 26 (GJB2) 2.86049 −0.57377 −0.09328 4.27E−04 mRNA,complete cds. /PROD = connexin 26 /FL = gb:NM_004004.1 gb:M86849.2 Homosapiens brain-derived 1.71599 −0.79412 0.616733 3.09E−02 neurotrophicfactor (BDNF), mRNA. /PROD = brain-derived neurotrophic factor /FL =gb:NM_001709.1 ESTs, Weakly similar to unknown 2.33677 −0.04066 1.52171.87E−03 (H. sapiens) STs 2.57265 0.177794 1.74876 2.74E−04 ESTs 2.68090.209095 1.70624 1.21E−02 Homo sapiens DKC1 gene, exons 1 to 4.183071.70283 3.20863 2.00E−02 11 Homo sapiens T cell receptor beta chain4.45228 1.38318 2.30031 3.27E−03 (TCRBV13S1-TCRBJ2S1) mRNA, completecds. /PROD = T cell receptor beta chain /FL = gb:AF043179.1 Homo sapiensPRO0066 mRNA, 0.466715 −1.87679 −0.22665 4.25E−03 complete cds. /PROD =PRO0066 /FL = gb:AF113007.1 axonal transport of synaptic vesicles5.20385 2.74124 4.27648 3.33E−03 Homo sapiens, Similar to cyclin M2,0.139313 −2.31405 −0.76333 1.46E−02 clone MGC: 12933 IMAGE:4308662,mRNA, complete cds. /PROD = Similar to cyclin M2 /FL = gb:BC021222.1Homo sapiens clone CDABP0095 3.87201 2.7936 5.73958 9.63E−03 mRNAsequence H. sapiens mRNA for ribosomal protein 2.00401 0.354674 2.730134.70E−04 L18a homologue. /PROD = ribosomal protein L18a homologue ESTs,Weakly similar to ALU7_HUMAN 3.43639 0.843826 2.28468 3.37E−02 ALUSUBFAMILY SQ SEQUENCE CONTAMINATION WARNING ENTRY (H. sapiens) Homosapiens, clone IMAGE:5242616, −0.525567 −2.07832 0.406964 2.23E−02 mRNA.Homo sapiens C1orf24 mRNA, complete 5.17968 2.58724 4.04213 1.98E−04cds. /PROD = C1orf24 /FL = gb:AF288391.1 gb:AB050477.1 gb:NM_022083.1Homo sapiens clone IMAGE:451939, 5.15776 2.22786 3.40438 2.60E−02 mRNAsequence Homo sapiens cDNA FLJ14942 fis, 0.682724 −2.08716 −0.740741.45E−02 clone PLACE1011185, highly similar to INSERTION ELEMENT IS1PROTEIN INSB. hypothetical protein FLJ20425 4.23689 1.90847 3.699881.15E−02 FL = gb:NM_017816.1 gb:AL136750.1 2.28308 −0.65903 0.5215868.87E−03 Homo sapiens matrix metalloproteinase 10 (stromelysin 2)(MMP10), mRNA. /PROD = matrix metalloproteinase 10 preproprotein /FL =gb:BC002591.1 gb:NM_002425.1 ESTs 3.67167 2.20619 4.86553 3.16E−03 Homosapiens prominin (mouse)-like 1 5.46002 2.79737 4.29571 1.38E−03(PROML1), mRNA. /PROD = prominin (mouse)-like 1 /FL = gb:NM_006017.1gb:AF027208.1 Homo sapiens mRNA; cDNA 2.40278 −0.68409 0.399469 1.48E−02DKFZp434M2415 (from clone DKFZp434M2415). Homo sapiens hypotheticalprotein 2.1471 0.312211 2.65409 2.28E−04 FLJ20701 (FLJ20701), mRNA./PROD = hypothetical protein FLJ20701 /FL = gb:NM_017933.1 Homo sapienscDNA FLJ35259 fis, 6.79033 3.86627 5.12737 1.23E−03 clone PROST2004251.Homo sapiens hypothetical protein 4.0369 1.34318 2.84336 7.69E−03FLJ39553 (FLJ39553), mRNA. /FL = gb:NM_173549.1 Humanmicrofibril-associated 3.89326 0.527618 1.35764 1.81E−03 glycoprotein-2MAGP-2 mRNA, complete cds. /PROD = microfibril- associatedglycoprotein-2 MAGP-2 /FL = gb:NM_003480.1 gb:U37283.1 Homo sapiensproprotein convertase 3.76897 0.368734 1.17396 1.38E−02 subtilisinkexintype 5 (PCSK5), mRNA. /PROD = proprotein convertase subtilisinkexin type5 /FL = gb:U56387.2 gb:NM_006200.1 Cluster Incl. AI735391:at10e09.x1Homo 2.59941 0.203564 2.03637 1.12E−04 sapiens cDNA, 3 end /clone =IMAGE− 2354728 /clone_end = 3 /gb = AI735391 /gi = 5056915 /ug =Hs.20137 /len = 567 ESTs 2.30031 −1.01596 −0.08964 1.46E−02 ruppel-likefactor 4 (gut) 3.50041 1.09954 2.95915 1.22E−02 Homo sapiens full lengthinsert cDNA −1.40946 −3.04877 −0.39378 3.32E−02 YI25A03 ESTs 0.806666−2.01081 −0.53076 1.91E−02 Homo sapiens mRNA; cDNA 3.1892 0.0127681.13562 1.35E−03 DKFZp761G02121 (from clone DKFZp761G02121); partial cdsHomo sapiens TMEFF2 mRNA, 2.28638 −0.89508 0.225546 1.21E−03 completecds. /FL = gb:AB017269.1 gb:NM_016192.2 gb:AF179274.2 gb:AF242222.1 Homosapiens, clone MGC:3328, 3.54625 0.772486 2.30168 4.16E−02 mRNA,complete cds. /PROD = Unknown (protein for MGC:3328) /FL = gb:BC001745.1gb:NM_014392.1 ESTs 2.18149 −0.58303 0.984522 2.68E−02 synaptojanin 24.166 1.83143 3.84199 0.00E+00 KIAA0367 protein 1.5999 −1.50555 −0.262861.58E−02 ESTs, Moderately similar to 0.868185 −1.46804 0.573078 1.69E−02ALU8_HUMAN ALU SUBFAMILY SX SEQUENCE CONTAMINATION WARNING ENTRY (H.sapiens) bromodomain adjacent to zinc finger 1.60872 −1.12544 0.5183612.11E−02 domain, 1B Human complement cytolysis inhibitor 6.61592 3.916655.596 1.66E−02 (CLI) mRNA, complete cds. /FL = gb:J02908.1gb:NM_001831.1 gb:M64722.1 gb:M25915.1 forkhead box O1A(rhabdomyosarcoma) 5.04975 2.59802 4.53545 5 1.60E−03 /FL =gb:NM_002015.2 gb:AF032885.1 gb:U02310.1 Homo sapiens cDNA: FLJ22727fis, 4.88391 2.09093 3.68875 1.91E−02 clone HSI15054 cadherin 6, type 2,K-cadherin (fetal 3.27371 0.243335 1.61199 2.74E−04 kidney) /FL =gb:D31784.1 gb:NM_004932.1 ESTs 3.97749 0.786845 1.99803 1.73E−02endonuclease G-like 1 1.11519 −2.09018 −0.88324 1.69E−02 FL =gb:AB020523.1 gb:NM_005107.1 Homo sapiens cDNA FLJ13034 fis, 4.596410.64328 1.1121 1.66E−02 clone NT2RP3001232 Human DNA sequence from cloneRP4- 2.91607 0.209555 1.92644 1.93E−02 61404 on chromosome 20q11.1-12Contains the 3 part of the MMP24 (matrix metalloproteinase 24(membrane-inserted)) gene, the ITGB4BP (integrin beta 4 binding protein)gene, the 3 end of a novel gene, the 3 end o . . . Homo sapiens,collapsin response 0.672753 −1.75265 0.250803 8.65E−03 mediatorprotein-5; CRMP3-associated molecule, clone MGC:11247, mRNA, completecds. /PROD = collapsin response mediator protein-5;CRMP3- associatedmolecule /FL = gb:BC002874.1 ESTs, Moderately similar to 2.915760.289656 2.10334 2.57E−03 ALU7_HUMAN ALU SUBFAMILY SQ SEQUENCECONTAMINATION WARNING ENTRY (H. sapiens) ESTs, Moderately similar toCA1C RAT 6.2681 1.94518 1.82747 4.12E−02 COLLAGEN ALPHA 1(XII) CHAIN(R.norvegicus) Homo sapiens alpha-aminoadipate 5.2604 2.72549 4.66854.33E−03 semialdehyde synthase mRNA, complete cds. /PROD = alpha-aminoadipate semialdehyde synthase /FL = gb:AF229180.1 Homo sapiens Wntinhibitory factor-1 1.03265 −1.51207 0.433159 8.62E−05 (WIF-1), mRNA./PROD = Wnt inhibitory factor-1 /FL = gb:AF122922.1 gb:NM_007191.1ribosomal protein S11 4.95358 2.25158 4.04683 2.98E−04 Homo sapienstumor necrosis factor, 2.42729 −0.73843 0.613222 1.93E−02 alpha-inducedprotein 6 (TNFAIP6), mRNA. /PROD = tumor necrosis factor, Iph-inducedprotein 6 /FL = gb:NM_007115.1 Homo sapiens hairyenhancer-of-split2.3848 −0.44154 1.27956 3.16E−03 related with YRPW motif 2 (HEY2), mRNA./PROD = hairyenhancer-of-split related with YRPW motif2 /FL =gb:NM_012259.1 gb:AF311884.1 gb:AB044755.1 gb:AF232238.1 gb:AF237949.1gb:AF173901.1 Homo sapiens semenogelin I (SEMG1), 0.585816 −2.46893−0.95223 3.05E−02 mRNA. /PROD = semenogelin I /FL = gb:J04440.1gb:NM_003007.1 Homo sapiens, Similar to cadherin 6, 2.38648 −0.022122.14359 2.59E−03 type 2, K-cadherin (fetal kidney), clone MGC:1470,mRNA, complete cds. /PROD = Similar to cadherin 6, type 2, K- cadherin(fetalkidney) /FL = gb:BC000019.1 Homo sapiens mRNA; cDNA 2.16899−0.56794 1.27546 3.16E−03 DKFZp434H0350 (from clone DKFZp434H0350);partial cds. /PROD = hypothetical protein Homo sapiens growth factorreceptor- 4.2346 1.17569 2.73613 4.80E−03 bound protein 14 (GRB14),mRNA. /PROD = growth factor receptor-bound protein 14 /FL = gb:L76687.1gb:NM_004490.1 Homo sapiens, Similar to TAF5-like RNA 2.10954 −0.13132.24853 8.24E−04 polymerase II, p300CBP-associated factor(PCAF)-associated factor, 65 kDa, clone MGC:46101 IMAGE:5551246, mRNA,complete cds. /PROD = Similar to TAF5-like RNA polymerase II,p300CBP-associated factor (PCAF)-associat Homo sapiens mRNA; cDNA 1.42404−1.99222 −0.77126 3.21E−02 DKFZp761J1324 (from clone DKFZp761J1324)solute carrier family 30 (zinc 2.82535 0.288493 2.38889 0.00E+00transporter), member 1 antizyme inhibitor 3.65887 0.37346 1.749121.86E−02 Homo sapiens GRO1 oncogene 2.51473 0.107263 2.38874 7.16E−04(melanoma growth stimulating activity, alpha) (GRO1), mRNA. /PROD = GRO1oncogene (melanoma growth stimulatingactivity, alpha) /FL =gb:NM_001511.1 Homo sapiens proprotein convertase 4.90999 0.8713091.52441 1.71E−02 subtilisinkexin type 5 (PCSK5), mRNA. /PROD =proprotein convertase subtilisinkexin type 5 /FL = gb:U56387.2gb:NM_006200.1 ESTs 0.254736 −1.96122 0.51777 4.00E−02 G-proteingamma-12 subunit 3.93653 1.06178 2.88889 5.11E−04 /FL = gb:NM_018841.1gb:AF119663.1 platelet-derived growth factor alpha 3.50343 0.6512462.50207 3.99E−03 polypeptide clusterin (complement lysis inhibitor, SP-2.88196 −0.42677 0.9712 8.48E−03 40,40, sulfated glycoprotein 2,testosterone-repressed prostate message 2, apolipoprotein J) ESTs2.82081 −1.69816 −1.89985 4.10E−02 Homo sapiens insulinoma-associated 12.94347 0.120494 2.02248 1.43E−03 (INSM1), mRNA. /PROD = insulinoma-associated 1 /FL = gb:NM_002196.1 gb:M93119.1 LBP protein 32 /FL =gb:NM_014552.1 2.68546 −0.16678 1.70626 1.87E−03 gb:AF198489.1 ESTs,Highly similar to T42654 2.84504 0.515703 2.95379 7.92E−04 hypotheticalprotein DKFZp434G1115.1 (H. sapiens) Homo sapiens Friend leukemia virus1.7744 −2.28064 −1.56765 3.14E−02 integration 1 (FLI1), mRNA. /PROD =Friend leukemia virus integration 1 /FL = gb:BC001670.1 gb:NM_002017.2gb:M98833.3 ESTs 0.846429 −2.42046 −0.91891 1.57E−02 Homo sapiensoxidative 3 alpha 1.30153 −1.64227 0.196394 3.20E−03 hydroxysteroiddehydrogenase; retinol dehydrogenase; 3-hydroxysteroid epimerase (RODH),mRNA. /PROD = oxidative 3 alpha hydroxysteroid dehydrogenase;retinoldehydrogenase; 3-hydroxysteroid epimerase /FL = gb:AF016509.1 gb:A minorhistocompatibility antigen HA-1 1.89498 −0.37136 2.15157 4.61E−02UDP-glucose pyrophosphorylase 2 4.26327 0.968939 2.4776 6.20E−03 Homosapiens regulator of G-protein 5.42952 3.05504 5.49711 1.21E−03signalling 2, 24 kD (RGS2), mRNA. /PROD = regulator of G-proteinsignalling 2, 24 kD /FL = gb:L13463.1 gb:NM_002923.1 ESTs 4.499871.01623 2.35659 1.86E−03 ESTs 0.623422 −1.94907 0.317138 8.95E−04 ESTs0.326084 −1.69499 1.13586 1.10E−02 ESTs 3.82952 0.673296 2.371123.92E−02 Homo sapiens cDNA FLJ10160 fis, 2.80876 −0.24405 1.568531.39E−02 clone HEMBA1003545, highly similar to INSULIN GENE ENHANCERPROTEIN ISL-2. thyroid hormone receptor-associated 4.66717 2.018834.2391 4.63E−03 protein, 150 kDa subunit /FL = gb:NM_005119.1gb:AF117756.1 2.4548 −0.3811 ESTs 1.65212 4.08E−03 Homo sapiens mRNA;cDNA 3.13835 0.605922 2.95511 1.53E−03 DKFZp667D095 (from cloneDKFZp667D095) Homo sapiens synaptojanin 2 mRNA, 2.94452 0.23595 2.41483.63E−04 complete cds. /PROD = synaptojanin 2 /FL = gb:AF318616.1 Homosapiens procollagen C- 3.37999 0.343624 2.21149 1.46E−02 endopeptidaseenhancer (PCOLCE), mRNA. /PROD = procollagen C- endopeptidase enhancer/FL = gb:BC000574.1 gb:NM_002593.2 b:AB008549.1 gb:L33799.1 3.244950.925809 3.52818 1.70E−04 ESTs Homo sapiens mRNA for KIAA0930 3.657720.911608 3.09513 8.62E−05 protein, partial cds. /PROD = KIAA0930 proteinHomo sapiens forkhead transcription 1.51993 −2.14186 −0.86243 3.75E−02factor FOXL2 (BPES), mRNA. /PROD = forkhead transcription factor FOXL2/FL = gb:AF301906.1 gb:NM_023067.1 ESTs 1.9867 −1.5948 −0.2171 2.81E−03ESTs, Highly similar to AF174600 1 F- 3.06175 0.202953 2.35691 2.45E−02box protein Fbx20 (H. sapiens) Homo sapiens ankyrin 3, node of 3.116440.101116 2.09865 2.90E−03 Ranvier (ankyrin G) (ANK3), transcript variant2, mRNA. /PROD = ankyrin 3, isoform 2 /FL = gb:NM_001149.1 gb:U43965.1Homo sapiens tumor necrosis factor 2.39086 −1.04254 0.558779 1.62E−02receptor superfamily, member 6 (TNFRSF6), mRNA. /PROD = apoptosis(APO-1) antigen 1 /FL = gb:NM_000043.1 gb:M67454.1 Homo sapiensthyrotropin-releasing 1.30849 −1.50688 0.822517 4.91E−02 hormonedegrading ectoenzyme (TRHDE), mRNA. /PROD = thyrotropin- releasinghormone degradingectoenzyme /FL = gb:AF126372.1 gb:NM_013381.1 ESTs4.65711 0.915563 2.32327 6.29E−03 Homo sapiens mRNA; cDNA 2.36072−2.02594 −1.25286 1.50E−02 DKFZp564N1116 (from clone DKFZp564N1116) Homosapiens placental protein 13-like 3.55894 0.434263 2.47085 3.48E−03protein (LOC56891), mRNA. /PROD = placental protein 13-like protein /FL= gb:NM_020129.1 gb:AF267852.1 Homo sapiens, Similar to receptor 2.055780.178992 3.5007 4.46E−04 tyrosine kinase-like orphan receptor 1, cloneMGC:12687, mRNA, complete cds. /PROD = Similar to receptor tyrosinekinase-like orphanreceptor 1 /FL = gb:BC006374.1 ESTs 0.159066 −2.96514−0.84976 3.25E−02 Homo sapiens, Similar to v-ets avian 2.22285 −1.58562−0.1074 2.63E−02 erythroblastosis virus E26 oncogene homolog 1, cloneMGC:29755 IMAGE:3946751, mRNA, complete cds. /PROD = Similar to v-etsavian erythroblastosis virusE26 oncogene homolog 1 /FL = gb:BC017314.1ESTs, Highly similar to dedicator of cyto- 0.83871 −2.29829 −0.129511.85E−02 kinesis 1 (H. sapiens) ESTs 1.1465 −1.7403 0.682768 3.99E−02ESTs 1.7466 −1.67509 0.283174 2.97E−03 ESTs, Weakly similar toPMXB_HUMAN 1.03192 −2.44485 −0.51457 1.87E−03 PAIRED MESODERM HOMEOBOXPROTEIN 2B (H. sapiens) ESTs 3.22222 −0.29339 1.59904 7.36E−03 Homosapiens cDNA FLJ10561 fis, 2.295 −1.60823 −0.08369 3.93E−03 cloneNT2RP2002672 Homo sapiens, Similar to KIAA0441 1.38545 −1.60426 0.869092.33E−03 gene product, clone MGC:45124 IMAGE:5578893, mRNA, completecds. /PROD = Similar to KIAA0441 gene product /FL = gb:BC036731.1 Homosapiens, clone IMAGE:4067166, 0.195869 −2.50507 0.276993 1.16E−03 mRNAESTs 1.5857 −2.61787 −1.30022 1.12E−02 Homo sapiens methyl-CpG binding0.490598 −3.45465 −1.70163 3.90E−03 protein MBD2 (MBD2) mRNA, completecds. /PROD = methyl-CpG binding protein MBD2 /FL = gb:NM_003927.2gb:AF072242.1 KIAA1151 protein 0.642527 −2.04288 1.00885 4.09E−03 H.sapiens mRNA for B-HLH DNA 5.43034 1.56039 3.45122 1.12E−04 indingprotein. /PROD = B-HLH DNA binding protein /FL = gb:NM_000474.1 Homosapiens, Similar to hypothetical 0.520711 −1.99011 1.26899 2.63E−02protein FLJ32001, clone MGC:39559 IMAGE:4828136, mRNA, complete cds./PROD = Similar to hypothetical protein FLJ32001 /FL = gb:BC036200.1Homo sapiens Wilms tumor 1 (WT1), 2.40462 −2.96375 −2.43546 1.41E−02transcript variant D, mRNA. /PROD = Wilms tumor 1 isoform D /FL =gb:NM_024424.1 gb:NM_024426.1 ESTs 1.53104 −1.54553 1.37309 8.14E−03Homo sapiens, Similar to cadherin 6, 2.54746 −0.69811 2.13419 1.45E−04type 2, K-cadherin (fetal kidney), clone MGC:1470, mRNA, complete cds./PROD = Similar to cadherin 6, type 2, K- cadherin (fetalkidney) /FL =gb:BC000019.1 ESTs 0.133612 −3.77953 −1.56636 1.40E−02 paternallyexpressed 3 2.87581 −1.67669 −0.0118 2.17E−02 Homo sapiens Charot-Leydencrystal 3.30086 −0.41644 2.1096 2.08E−02 protein (CLC), mRNA. /PROD =Charot- Leyden crystal protein /FL = gb:NM_001828.3 gb:L01664.1 ESTs3.26198 −0.72777 1.5682 3.16E−03 Homo sapiens tachykinin, precursor 12.77265 −1.02081 1.48319 4.07E−04 (substance K, substance P, neurokinin1, neurokinin 2, neuromedin L, neurokinin alpha, neuropeptideK,neuropeptide gamma) (TAC1), transcript variant beta, mRNA. /PROD =tachykinin 2 precursor, isoform beta /FL = gb:U3 Homo sapiens PNAS-123mRNA, 1.59659 −1.46656 1.84663 3.90E−03 complete cds Homo sapienshypothetical protein −0.359245 −3.46654 −0.06528 2.55E−03 FLJ20075(FLJ20075), mRNA. /PROD = hypothetical protein FLJ20075 FL =gb:NM_017655.1 Human platelet-derived growth factor 2.44464 −0.58462.90484 3.63E−04 alpha-receptor (PDGFRA) mRNA, exons 13-16 Homo sapienscDNA: FLJ22547 fis, 4.29365 −0.10882 2.09548 1.54E−02 clone HSI00356Homo sapiens HSPC156 protein 0.786188 −2.32161 1.29619 8.62E−05(HSPC156), mRNA. /PROD = HSPC156 protein /FL = gb:NM_014178.1gb:AF161505.1 Homo sapiens mRNA; cDNA 0.485642 −4.8524 −3.09356 2.02E−02DKFZp586P1124 (from clone DKFZp586P1124) caldesmon 1 /FL = gb:M64110.17.09236 2.67863 5.63436 7.47E−03 gb:NM_004342.2 Homo sapiens, Similar tohypothetical 1.69808 −2.74939 0.215566 1.92E−02 protein FLJ10058, cloneMGC:34305 IMAGE:5167647, mRNA, complete cds. /PROD = Similar tohypothetical protein FLJ10058 /FL = gb:BC034293.1 Homo sapiens, Similarto LIM homeobox −0.062884 −4.30121 −1.10961 1.16E−02 protein 8, cloneIMAGE:4839343, mRNA. Homo sapiens chorionic 0.812166 −2.21846 2.218433.60E−03 somatomammotropin hormone 2 (CSH2), transcript variant 4, mRNA./PROD = chorionic somatomammotropin hormone 2, isoform4 /FL =gb:NM_022646.1 Homo sapiens HES-related repressor 2.80599 −1.193962.40562 2.44E−03 protein 1 HERP1 mRNA, complete cds. /PROD = HES-relatedrepressor protein 1 HERP1 /FL = gb:NM_012259.1 gb:AF311884.1gb:AB044755.1 gb:AF232238.1 gb:AF237949.1 gb:AF173901.1 Homo sapienscadherin 6, type 2, K- 0.824752 −3.83509 −0.87634 2.49E−02 cadherin(fetal kidney) (CDH6), mRNA. PROD = cadherin 6, type 2, K-cadherin(fetal kidney) /FL = gb:D31784.1 gb:NM_004932.1 paternally expressed 32.87398 −4.12925 −3.49232 2.19E−02 Homo sapiens cDNA FLJ11398 fis,2.5268 −1.71619 1.84868 4.46E−04 clone HEMBA1000637. Homo sapiensBCL2-interacting killer 4.30042 0.023414 3.56237 1.94E−03(apoptosis-inducing) (BIK), mRNA. /PROD = BCL2-interacting killer /FL =gb:NM_001197.2 gb:BC001599.1 gb:U34584.1 gb:U49730.1 Homo sapiens testisexpressed 3.51154 −2.84891 −1.3824 2.18E−02 sequence 14 (TEX14), mRNA./PROD = testis expressed sequence 14 /FL = gb:NM_031272.1 Homo sapiensgrowth hormone 2 (GH2), 0.922569 −2.77587 1.4679 1.60E−03 transcriptvariant 2, mRNA. /PROD = growth hormone 2, isoform 2 precursor /FL =gb:J03756.1 gb:NM_022557.1 Homo sapiens, clone IMAGE:4828836, 0.972907−4.24111 −1.48398 1.63E−02 mRNA. Homo sapiens galanin receptor 1 2.50745−3.13141 −0.33802 3.38E−03 (GALR1), mRNA. /PROD = galanin receptor 1 /FL= gb:NM_001480.2 gb:U23854.1 gb:L34339.1 gb:U53511.1 Homo sapiens,serine (or cysteine) 1.96851 −4.13943 −1.56568 3.06E−02 proteinaseinhibitor, clade B (ovalbumin), member 3, clone MGC: 12244, mRNA,complete cds. /PROD = serine (or cysteine) proteinase inhibitor, cladeB(ovalbumin), member 3 /FL = gb:NM_006919.1 gb:U19556.1 gb:BC005224.1DKFZP566K1924 protein 2.88225 −2.59632 1.39062 0.00E+00 ESTs, Moderatelysimilar to 4.24074 −0.69782 3.87444 1.12E−04 ALU2_HUMAN ALU SUBFAMILY SBSEQUENCE CONTAMINATION ARNING ENTRY (H. sapiens) Human mRNA upregulatedduring 0.494505 −4.62912 −0.18915 4.38E−02 camptothecin-inducedapoptosis of U937 cells. nuclear receptor subfamily 1, group I, 1.77902−4.02806 −0.19682 4.34E−02 member 3 ESTs −1.3795 −6.08172 −0.946066.91E−04 Homo sapiens chorionic 1.12437 −3.12138 2.61605 1.12E−04somatomammotropin hormone 2 (CSH2), transcript variant 1, mRNA. /PROD =chorionic somatomammotropin hormone 2, isoform 1precursor /FL =gb:NM_020991.2 gb:BC002717.1 Homo sapiens mRNA for SCCA2b, 3.32911−3.16288 0.568045 1.93E−02 complete cds. /PROD = SCCA2b /FL =gb:AB046400.1 Homo sapiens KIAA0469 gene product 4.03097 −1.948262.56518 2.49E−04 (KIAA0469), mRNA. /PROD = KIAA0469 gene product /FL =gb:AB007938.1 gb:NM_014851.1 ESTs 4.45389 −2.40636 5.33359 5.65E−04

TABLE VII THE EFFECT OF WNT-3A TREATMENT ON CYTOKINE EXPRESSION INPOPULATIONS OF CELLS FROM THE HUMAN EMBRYONIC STEM CELL LINE H9. Celllysate Cell conditioned media w/o wnt 1 w/o wnt 2 w/ wnt 1 w/ wnt 2 w/ownt 1 w/o wnt 2 w/ wnt 1 w/ wnt 2 POS 62,842.33 67,606.06 49,758.3450,702.57 96,585.15 109,721.82 199,709.04 195,889.94 NEG 23.38 34.49370.82 400.66 58.06 34.45 216.90 105.20 Angiogenin 39.50 132.91 619.40750.85 13,726.35 11,749.55 57,442.48 54,969.04 BDNF 167.50 167.82 763.51951.45 689.22 563.39 770.06 715.85 BLC 2.00 48.99 742.24 476.36 107.69512.77 337.12 249.80 BMP-4 247.00 273.70 348.44 304.48 583.40 541.831,156.54 771.19 BMP-6 8.50 141.92 571.23 641.05 354.91 355.28 985.70670.60 CK beta 8-1 1.00 1.13 22.48 21.54 17.79 25.31 42.78 75.44 CNTF1.00 55.19 1,382.51 1,060.62 282.81 211.86 787.19 541.51 EGF 32.50 82.22742.24 984.39 1,681.85 1,406.12 23,331.57 22,768.53 Eotaxin 1.00 7.32301.87 253.80 207.89 199.67 622.91 291.71 Eotaxin-2 262.00 336.771,155.31 960.31 890.56 744.31 2,156.21 1,445.14 Eotaxin-3 184.50 284.96934.12 847.56 975.77 665.56 2,140.81 1,480.35 FGF-6 629.00 379.571,327.92 964.54 1,239.85 947.73 3,283.94 2,519.78 FGF-7 31.00 222.451,075.02 941.73 449.49 352.47 1,086.66 861.72 Fit-3 Ligand 44.00 180.21857.45 795.19 315.58 277.47 511.67 792.98 Fractalkine 23.50 62.51 792.02456.51 120.80 145.30 359.37 367.15 GCP-2 1.00 45.05 593.71 538.86 203.21166.86 451.78 362.12 GDNF 84.00 292.84 436.75 444.68 176.99 194.98443.22 509.66 GM-CSF 433.00 636.37 1,343.17 1,161.75 777.25 722.751,808.82 1,446.82 I-309 152.50 246.10 708.52 743.25 1,828.87 179.05318.30 288.35 IFN-gamma 349.00 441.52 1,202.67 1,037.60 283.74 559.641,238.97 1,435.08 IGFBP-1 1.50 3.38 59.41 85.73 33.71 24.37 227.60155.91 IGFBP-2 3,785.50 5,554.45 2,671.90 2,414.72 27,656.85 28,473.9542,215.53 43,934.33 IGFBP-4 5,553.00 6,043.27 4,459.85 4,173.1910,091.09 6,029.45 18,986.34 13,360.14 IGF-I 1.00 2.25 558.38 208.1988.96 148.11 254.98 187.77 IL-10 158.00 422.93 1,222.75 1,096.30 722.93564.32 1,625.71 1,129.96 IL-13 428.50 555.84 1,486.89 1,407.96 1,114.36786.49 2,522.42 1,807.27 IL-15 599.50 808.13 1,677.16 1,601.79 1,140.59858.67 2,495.04 1,981.62 IL-16 2.00 137.97 651.11 668.50 259.39 214.67436.36 405.71 IL-1alpha 1,236.00 1,216.99 1,612.93 1,421.89 10,155.705,842.90 3,999.26 3,545.79 IL-1beta 1.00 1.13 352.05 312.08 117.99 75.93316.59 278.30 IL-1ra 33.00 25.91 302.69 198.48 142.34 93.74 364.23581.75 IL-2 210.50 681.99 966.23 1,133.88 188.22 363.72 756.10 861.84IL-3 3,146.50 1,976.13 2,119.94 1,497.48 4,598.86 5,260.77 11,345.7812,174.73 IL-4 17.50 74.34 625.42 636.94 357.72 239.04 804.30 673.95IL-5 400.50 555.84 1,245.23 1,167.24 1,056.31 791.18 2,120.27 1,713.38IL-6 628.50 516.98 1,121.19 842.07 7,377.28 9,527.86 9,774.82 9,991.93IL-7 190.00 313.12 1,212.31 531.66 456.98 430.27 681.09 1,017.63 Leptin900.50 617.79 1,234.39 1,091.65 2,999.42 2,016.38 7,236.71 4,886.99LIGHT 10.50 232.02 1,081.04 888.10 619.92 453.71 1,346.78 1,108.17 MCP-1242.50 262.71 1,022.03 1,076.03 572.17 500.58 2,015.89 1,567.53 MCP-224.00 109.25 909.63 1,152.46 129.23 351.53 694.78 694.07 MCP-3 1.00168.38 1,184.61 1,255.50 401.73 169.67 701.62 583.42 MCP-4 7.50 46.741,380.51 1,608.86 123.61 61.87 231.02 276.62 M-CSF 1.00 63.64 852.63867.83 529.09 269.04 740.98 684.01 MDC 1.00 2.25 269.36 260.41 157.3252.50 280.65 132.44 MIG 291.00 226.39 1,113.16 1,137.26 272.50 486.52617.77 1,222.17 MIP-1-delta 1.50 1.13 491.75 322.22 144.21 66.56 474.02291.71 MIP-3-alpha 1.00 5.63 1,602.09 1,399.93 55.25 0.00 208.78 164.41NAP-Z 1.00 6.19 465.66 177.37 97.39 110.61 371.35 343.66 NT-3 1.00 1.13316.73 275.76 97.39 171.55 571.57 427.51 PARC 1.00 2.25 352.85 353.04170.43 81.56 474.02 363.80 PDGF-BB 437.00 1,644.99 517.84 597.564,729.96 8,521.09 2,635.37 2,005.09 RANTES 1.00 89.54 724.98 637.25393.31 347.78 682.80 855.01 SCF 74.50 148.67 1,152.50 1,021.13 691.09487.46 1,394.69 2,155.98 SDF-1 23.50 51.25 940.14 1,294.78 362.07 123.74326.85 616.95 TARC 1.50 5.07 372.93 343.33 206.95 71.24 277.23 1,642.97TGF-beta 1 362.00 230.33 1,426.67 1,240.30 752.90 740.56 1,827.651,849.18 TGF-beta 3 3.00 25.91 377.74 1,130.93 111.44 115.30 294.34343.68 TNF-alpha 431.00 579.49 2,851.74 1,285.91 1,205.20 929.912,609.70 2,167.71 TNF-beta 241.00 342.40 1,049.33 1,026.62 675.17 671.191,701.01 1,637.94 Internal Control 18,642.67 18,642.67 18,642.6718,642.67 18,642.67 18,642.67 18,642.67 18,642.67 POS 60,794.8348,106.85 35,652.42 47,391.58 94,013.71 90,889.16 141,321.30 149,125.43NEG 11.13 5.53 11.22 30.45 24.93 17.75 36.22 33.27 Acrp30 1,036.00744.50 601.61 690.66 1,797.52 1,319.68 2,795.71 2,302.99 AgRP 11.00153.99 77.82 89.00 378.17 195.90 651.26 482.21 Angiopolatin-2 28.5010.43 357.97 771.04 655.82 632.60 65,959.24 71,664.21 Amphiregulin 17.5015.44 73.63 154.62 250.52 151.01 619.07 762.60 AxI 637.50 795.93 88.59139.03 401.31 324.05 610.40 748.34 bFGF 59,820.50 54,057.29 11,722.9521,398.52 7,948.03 4,993.63 1,226.99 1,241.09

1. A method for producing pancreatic endoderm lineage cells expressingNKX2.2 and PDX1, comprising: obtaining definitive endoderm cells; anddifferentiating the definitive endoderm cells with a medium comprising atransforming growth factor (TGF)-β receptor agonist, retinoic acid or aretinoic acid analog, and a fibroblast growth factor (FGF), therebyproducing pancreatic endoderm lineage cells expressing NKX2.2 and PDX1.2. The method of claim 1, wherein the TGF-β receptor agonist is activinA.
 3. The method of claim 2, wherein the medium comprises 100 ng/mLactivin A.
 4. The method of claim 1, wherein the medium comprises 1 μMretinoic acid
 5. The method of claim 1, wherein the FGF is FGF-2, FGF4or FGF-10.
 6. The method of claim 5, wherein the FGF is FGF-2.
 7. Themethod of claim 6, wherein the medium comprises 50 ng/mL FGF-2.
 8. Themethod of claim 1, wherein obtaining definitive endoderm cells comprisesdifferentiating pluripotent stem cells into definitive endoderm cells bytreating the pluripotent stem cells with a TGF-β receptor agonist. 9.The method of claim 8, wherein the TGF-β receptor agonist is activin A.10. A composition comprising an isolated human pancreatic endodermlineage cell population expressing NKX2.2 and PDX1, a transforminggrowth factor (TGF)-β receptor agonist, retinoic acid and a fibroblastgrowth factor (FGF).
 11. The composition of claim 10, wherein the TGF-βreceptor agonist is activin A.
 12. The composition of claim 11,comprising 100 ng/mL activin A.
 13. The composition of claim 10,comprising 1 μM retinoic acid
 14. The composition of claim 10, whereinthe FGF is FGF-2, FGF4 or FGF-10.
 15. The composition of claim 14,wherein the FGF is FGF-2.
 16. The composition of claim 15, comprising 50ng/mL FGF-2.